Article pubs.acs.org/JPCB
Bidirectional Photoinduced Electron Transfer in Ruthenium(II)-Trisbipyridyl-Modified PpcA, a Multi-heme c‑Type Cytochrome from Geobacter sulf urreducens Oleksandr Kokhan,†,# Nina S. Ponomarenko,†,§ P. Raj Pokkuluri,‡ Marianne Schiffer,‡ Karen L. Mulfort,† and David. M. Tiede*,† †
Chemical Sciences and Engineering Division and ‡Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, Illinois 60439, United States S Supporting Information *
ABSTRACT: PpcA, a tri-heme cytochrome c7 from Geobacter sulf urreducens, was investigated as a model for photosensitizerinitiated electron transfer within a multi-heme “molecular wire” protein architecture. Escherichia coli expression of PpcA was found to be tolerant of cysteine site-directed mutagenesis, demonstrated by the successful expression of natively folded proteins bearing cysteine mutations at a series of sites selected to vary characteristically with respect to the three -CXXCH- heme binding domains. The introduced cysteines readily reacted with Ru(II)-(2,2′-bpy)2(4-bromomethyl-4′-methyl-2,2′-bipyridine) to form covalently linked constructs that support both photooxidative and photo-reductive quenching of the photosensitizer excited state, depending upon the initial heme redox state. Excited-state electron-transfer times were found to vary from 6 × 10−12 to 4 × 10−8 s, correlated with the distance and pathways for electron transfer. The fastest rate is more than 103-fold faster than previously reported for photosensitizer−redox protein constructs using amino acid residue linking. Clear evidence for inter-heme electron transfer within the multi-heme protein is not detected within the lifetimes of the charge-separated states. These results demonstrate an opportunity to develop multi-heme ccytochromes for investigation of electron transfer in protein “molecular wires” and to serve as frameworks for metalloprotein designs that support multiple-electron-transfer redox chemistry.
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INTRODUCTION Protein “molecular wire” architectures found in the dissimilatory metal-reducing bacteria Geobacter and Shewanella are of widespread interest as models for long-range electron transfer within multiple cofactor proteins1,2 and for resolving the molecular basis for microbial respiration through extra-cellular redox chemistry.1,3−5 Physiologically, the multi-heme ccytochromes are described to function as molecular wires and “capacitors” for the storage and rapid discharge of multiple reducing equivalents in environmental metal-oxide and sulfatereducing microbial metabolism, span a redox potential range from −100 to −360 mV (vs NHE) in different protein homologues, function as combined electron and proton (redox Bohr) donors, and pack a high density of heme cofactors within small protein domains that typically have about 1 heme group per 25 amino acid residues.5−10 Electron delocalization among the hemes in the cytochromes c7 has been demonstrated to be rapid on an NMR time scale,11−13 suggestive of the molecular wire and electron storage functions.5,6 Three-heme domains of cytochrome c7 architectures have been structurally characterized.11−21 As such, the cytochromes c7 are well-suited to serve as models to investigate electron transfer in protein molecular © XXXX American Chemical Society
wire architectures and to serve as frameworks for metalloprotein designs that support multiple-electron-transfer redox chemistry. Mechanisms for electron transfer within redox proteins have been extensively investigated by labeling with ruthenium and rhenium coordination complexes that possess long-lived, metalto-ligand charge-transfer (MLCT) excited states for photoinitiated electron transfer.22−26 For c-type cytochromes, photoinduced electron transfers have been initiated directly from the MLCT excited-state and flash-quenched ground states using ruthenium and rhenium complexes coordinated to histidine residues,27−30 and with functionalized ruthenium polypyridine complexes covalently linked to lysine,31,32 and cysteine33−35 residues. Pertinent as benchmarks for the results presented here, we note the fastest rates for photo-induced electron transfer between a photosensitizer linked to an amino acid residue and a redox protein cofactor have been reported for Special Issue: John R. Miller and Marshall D. Newton Festschrift Received: November 18, 2014 Revised: February 26, 2015
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allowing either excited-state electron or hole injection into PpcA, depending upon the initial oxidation state of the heme cofactors. Excited-state electron-transfer rates vary more than 4 orders of magnitude depending on the position of photosensitizer attachment, with the fastest electron-transfer rates occurring on the picosecond time scale. Further, results from molecular dynamics (MD) simulations indicate that for the two mutants with the fastest charge transfer rates, A23C and K29C, the photo-induced electron-transfer rates vary by a factor of 20, and can be correlated with specific paths for electron transfer.
ruthenium(II)-bis(bipyridine)-dicarboxybipyridine complexes linked to lysine residues that are positioned in close proximity to the exposed heme edge in cytochrome c, and are found to support electron-transfer rates up to 2 × 107 s−1 from the MLCT state and 3 × 107 s−1 for charge recombination.32 Faster rates for photo-induced electron transfer have been observed for photosensitizers directly coordinated to heme cofactors. For example, nitric oxide synthase coordinated with an imidazole functionalized rhenium-wire complex demonstrated a 6 × 108 s−1 rate of electron transfer to heme following reductive quenching of the MLCT state by tryptophan oxidation.36,37 Rates for photosensitized electron transfer in redox proteins are generally found to correlate with through-bond pathways between the photosensitizer and heme cofactor.23−27 The molecular wire or shuttle function of c-cytochromes is based on the ability to support efficient bi-directional electron transfer. Electrostatically stabilized complexes between Znporphyrin-substituted cytochrome c and a hemicarcerand were shown to support bidirectional excited-state electron transfer depending upon whether small electron donors or acceptors guest molecules were added to the hemicarcerand.38 Recently, bidirectional excited-state electron transfer was reported for the electrostatically stabilized complex formed between Mn- and Zn-porphyrin-substituted myoglobin and cytochrome c peroxidase that was measured by following the photosensitizer excited-state decays,39 although transient spectra show a minimal accumulation of a transient charge-separated state.39,40 In this report we describe our results on attempts to introduce cysteine mutations into PpcA, a three-heme c7-type cytochrome from Geobacter sulf ureducens, Figure 1, and
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EXPERIMENTAL SECTION Ru(bpy)2(4-bromomethyl-4′-methyl-2,2′-bipyridine)·2PF6, Ru(bpy)2(Br-bpy), was synthesized following a previously published procedure.41 1H NMR was performed on a Bruker DMX500 and referenced to TMS or residual solvent peak. ESIMS was collected on a ThermoFisher LCQ Fleet, from dilute acetonitrile solution in positive ionization mode. All characterization results matched previously reported values. Cysteine mutations in PpcA were introduced to template pVA203 plasmid,16 using the Stratagene QuickChange II mutagenesis kit and primer oligonucleotides (Sigma-Aldrich) summarized in the Supporting Information, Table S1, and following the manufacturer’s protocol. Mutant DNA sequences were verified at University of Chicago DNA Sequencing and Genotyping Facility. Mutant forms of PpcA were expressed in BL21 (DE3) Escherichia coli strain containing cytochrome c maturation genes (Ccm) and purified as described previously,42 with an additional step involving reduction of the engineered cysteine residues with 2 mM DTT in 10 mM Tris, pH 7.5, and 100 mM NaCl for 2 h at room temperature followed by gel filtration on a Sephadex G25 column. The molecular mass for all of the expressed cysteine mutants, as well as the products following Ru(bpy)2(Br-bpy) labeling described below, were analyzed using LC/ESI-MS. The liquid chromatography used a 2−30% MeCN linear gradient in water with 0.1% formic acid on C18 Discovery Bio analytical HPLC column (Sigma-Aldrich) installed on an Acella HPLC system (ThermoFisher), and analyzed with a diode array detector and ESI-MS (LCQ Fleet, ThermoFisher). The molecular weights of the purified cysteine PpcA mutants were found to consistently exceed the value expected from the protein sequence plus the contribution of three covalently attached hemes. After treatment with reducing agents like DTT or 2-mercaptoethanol, we observed the apparent molecular weight of the protein to shift to the values expected for each of the mutant forms, Supporting Information, Figure S1. We attribute this effect to a reversible binding of E. coli metabolites to engineered cysteine residues. This observation prompted us to add an additional step in our PpcA purification procedure where DTT treated protein was re-purified on a Sephadex G25 column described above. Labeling of reduced cysteine mutants of PpcA (0.5−1 mM) with Ru(bpy)2(Br-bpy) followed procedures described for labeling other redox proteins using this reagent.43,44 Here, the labeling of mutant PpcA was performed at room temperature in 10 mM Tris, pH 7.5 buffer with 100 mM NaCl with a 5-fold molar excess of Ru(bpy)2(Br-bpy). After overnight gentle shaking in a vial shielded from light, samples were centrifuged to pellet particulates and the supernatant was applied to a Sephadex G25 column to separate unreacted labeling reagent from the PpcA protein. A red cytochrome band showed a clear separation from an orange band of unreacted Ru(bpy)2(Brbpy). The covalent attachment of the label to the protein was
Figure 1. Tri-heme PpcA cytochrome from G. sulfurreducens (1) and Ru(II)-(2,2′-bpy)2(4-bromomethyl-4′-methyl-2,2′-bpy) (2) used for labeling PpcA at cysteine sites. The PpcA structure shows the heme I, III, IV nomenclature and locations selected for site-directed cysteine mutagenesis.
covalently attach a Ru(bpy)3 photosensitizer at selected locations in close proximity to the heme cofactors, including those with distance separations shorter than those reported todate. This work provides the first demonstration of photosensitizer-initiated, excited-state electron transfer within a multi-heme redox protein framework, and establishes a foundation for investigating long-range, multi-electron transfer within protein wire motifs. Of particular interest are possible applications that use multi-heme c-cytochromes to serve as sources/sinks for multi-electron catalysis in hybrid assemblies constructed with photosensitizer−catalyst dyad assemblies. The results show capabilities for bidirectional electron transfer, B
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B shown in Figure 2. Excitation of Ru(II)(bpy)3 with 460 nm light initially produces a singlet MLCT state (not shown),
verified with HPLC-MS as described above. Following the linkage reaction, we observed shifts in the position of chromatographic peaks, Supporting Information, Figure S2, and the expected shift in molecular weight of the protein due to covalent attachment of Ru(bpy)3, Supporting Information, Figure S3. Optical absorption spectra for Ru(bpy)3-labeled PpcA constructs show that the spectra are the sum of Ru(bpy)3 and PpcA optical absorptions, with no discernible alteration to the optical absorption of either Ru(bpy)3 or PpcA hemes. This is shown by comparison of the spectra for K29C, the K29C-Ru construct, the Ru(bpy)2(Br-bpy) label, and the summed K29C plus Ru(bpy)2(Br-bpy) spectrum, Supporting Information, Figure S4. Comparable spectra were recorded for each of the other mutant constructs. All-atom MD simulations were performed using computational resources of Laboratory Computing Resource Center (Argonne National Laboratory, Lemont, IL). Initial atom coordinates were taken from the 2LDO structure of PpcA.45 In silico mutations were introduced using VMD46 and the system with an explicit water box was allowed to equilibrate for 10 ns in all-atom MD simulations performed with NAMD2.47 This step was followed by addition of covalently linked Ru(bpy)2(Cys-bpy) photosensitizers and simulations were continued with the parameters as described previously.42 Force field parameters for Ru(bpy)2(Cys-bpy) were developed following the standard guidelines for CHARMM force field. For both K29C and A23C mutants, three independent ∼90 ns long simulation were performed. The intermediate structural snapshots were recorded each 10 ps and were analyzed with VMD. Sequences of all PDB files available as of January 2013 in the Protein Data Bank (PDB, www.rcsb.org/pdb) were scanned with a Tcl/TK script for simultaneous occurrences of c-type heme binding motifs (CXXCH) and at least one additional cysteine residue not belonging to CXXCH sequence. All structures meeting this criterion were loaded to VMD and the presence of covalently attached hemes was verified with another Tcl/Tk script. All proteins meeting both this criteria were manually inspected in order to confirm the presence of at least one c-type heme as well as the presence and structural location of any additional free cysteine residues. Proteins which had additional cysteine residues only in the form of disulfide bridges or heme axial ligands were excluded from the final results summarized in the Supporting Information, Table S2. Small- and wide-angle X-ray scattering (SAXS/WAXS) experiments were performed at the 12-ID-B beamline of the Advanced Photon Source at Argonne National Laboratory. Protein samples in 50 mM Tris buffer, pH 7.5 with 100 mM NaCl were concentrated to yield final protein concentration in the range of 0.2−0.5 mM. To minimize protein damage by Xray radiation, samples were gently refreshed in a flow cell with a syringe pump. All other parameters of combined SAXS/WAXS data collection were the same as described previously.42 Time-resolved transient absorbance measurements were performed using a Ti:sapphire-pumped OPA laser system developed at the Center for Nanomaterials, Argonne National Laboratory (http://www.anl.gov/cnm/nanophotonicscapabilities), and having an output pulse with a 120 fs halfwidth tuned to 460 nm for excitation of the Ru(bpy)3 photosensitizer as described previously.48 Samples were maintained in a nitrogen-purged atmosphere. Transient absorption kinetics for the Ru(bpy)3-linked PpcA derivatives were analyzed using the photochemical reaction schemes A and
Figure 2. Photochemical reaction schemes for Ru(bpy)3-labeled PpcA constructs. Schemes A and B outline a sequence of photochemical states starting with the PpcA hemes either in the ferric or ferrous redox states, respectively. Other aspects are described in the text. The Fe(III) and Fe(II) oxidation states for the PpcA hemes are colored red and blue, respectively, for emphasis.
which decays rapidly to a luminescent triplet MLCT state,49,50 indicated by 3[Ru(III)L−] in schemes A and B. The 3[Ru(III)L−] state can spontaneously decay to the ground state, kd, or undergo charge separation, kCS, followed by charge recombination, kCR. Schemes A and B in Figure 2 outline the photochemical states starting with the PpcA hemes either in the ferric or ferrous redox states, respectively. In the following text, 3 [Ru(III)L−] is abbreviated as *Ru(II). Time-dependent decay of the MLCT state, *Ru(II), and population of the charge-separated (CS) state within a threestate photochemical reaction scheme follow eqs 1 and 2, respectively.32,39 [*Ru(II)](t ) = Ao[e−(kCS+ kd)t ]
(1)
[CS](t ) = A[e−(kCS+ kd)t − e−kCRt ]
(2)
Here CS refers to the charge-separated states Fe(II)−Ru(III)L and Fe(III)−Ru(II)L− shown in schemes A and B in Figure 2, respectively. In the transient absorption measurements the population of the charge-separated state was monitored by following the optical absorption of the hemes using the transient absorption difference 552 nm − 541 nm. Here, 541 nm is an isosbestic point for redox changes in the heme,51 and its use as a reference wavelength allows the broad transient optical absorption changes arising from the ruthenium photosensitizer excited- and redox-state transitions to be subtracted from the heme transient kinetics. The heme transient absorption kinetics were fit using eq 2 with the biexponential time constants k1 = (kCS + kd) and k2 = kCR, and using the Levenberg−Marquardt algorithm in Origin 9.1. As discussed below, under conditions kCS ≫ kd, the rate constant fit is k1 ≈ kCS.
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RESULTS Protein Expression and Photosensitizer Labeling with Site-Selected Cysteine Mutations. Cytochromes c are characterized by covalent attachment of heme cofactors through two thioether linkages between heme vinyl groups and cysteine residues in proteins having the -CXXCH- (-CysC
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residue conservation
approximate distance Ru(bpy)3 to closest hemea
characteristics of mutation site
A23C K29C M45C
weak weak weak
K7C
weak
residue located in an α helix and forming van der Waals contact with one of the propionates of heme III within the CXXCH binding domain for heme III: CK-K29C-CH residue located in helix with Sγ atom of the engineered residue expected to be approximately the same in all three hemes linker sequence connecting two β sheets
E39C
moderate
residue located in coiled structure
E56Cb K70Cb
weak high
residue located in a helix and immediately following His residue in the CXXCH binding motif for heme III residue immediately preceding C-terminus and immediately following His residue of the CXXCH binding motif responsible for heme IV attachment
6.0 Å, Bpy to heme III 6.8 Å, Bpy to heme III 9 Å, Sγ atom of C-Ru to hemes I, II, IV 10 Å, Sγ atom of C-Ru to heme IV 11 Å, Sγ atom of C-Ru to heme I
a
Distance estimate is the closest edge-to-edge approach between conjugated atoms on the bipyridyl ligands and those on the heme cofactor. bThe bottom two rows are for cysteine mutants not used for Ru(bpy)2(Br-bpy) labeling in this report.
Xaa-Xaa-Cys-His-) amino acid sequence motif.51 There are relatively few examples of c-type cytochromes in the Protein Data Bank,52 with Cys residues not involved in heme binding. A comprehensive search revealed only 10 examples of distinct families of c-type cytochromes having additional cysteines not involved in covalent linkage to the heme cofactor, formation of disulfide bridges, or axial ligation of the heme iron atom, Supporting Information, Table S2. This limited set of natural examples did not reveal any obvious trends in position and secondary structure requirements for placement of additional free cysteine residues. Furthermore, 9 out of 10 families of such proteins had a substantially higher ratio of amino acids per ctype heme (100 or more) than the ratios typical for cyt c7 family (23−25 residues per heme). Therefore, it was difficult to speculate on possible constraints for the introduction of free Cys within c-type cytochromes. However, Allen and co-workers previously demonstrated that the Ccm apparatus in E. coli correctly recognized and attached heme when c-type sequences were introduced into cyt b562, including those that introduced Cys adjacent (CCXXCH, CXXCHC) or within (CCXCH, CXCCH) the c-type heme binding motif.53 These results demonstrate that the maturation enzymes responsible for covalently linking of heme to proteins bearing the cytochrome c consensus sequence are likely to be tolerant of adjacent free cysteine residues, but this study did not attempt to assay or reproduce the native cyt b562 protein fold. Protein sequence alignments for tri-heme-containing cytochromes showed that PpcA has a substantial number of highly conserved residues, corresponding to approximately 44% of the 71 amino acid sequence, Supporting Information, Table S3. Currently, it is not clear whether these play structural or physiological roles, or how tolerant the conserved sites are to amino acid mutation. For initial labeling studies in PpcA, we introduced mutations for Cys residues in 7 different positions that sampled a range of structural and sequence conditions, Figure 1 and Table 1, but primarily from the weakly conserved regions of the protein. From 7 mutants with verified DNA sequences, we found that the first 6 mutations listed in Table 1 expressed PpcA in amounts comparable to wild-type protein, while K70C mutant showed a markedly lower expression level. Labeling studies for the first 5 of 7 cysteine mutations listed in Table 1 were carried out using the Ru(bpy)2(Br-bpy) ligand. In each case, LC/ESI-MS analyses demonstrated quantitative labeling of introduced cysteine residues in PpcA mutants using the Ru(bpy)2(Br-bpy) ligand. Combined SAXS/WAXS was used to examine possible alternation of the PpcA protein fold
due to cysteine mutations and following Ru(bpy)3 covalent attachment. Previous studies demonstrated that misfolding and errors in heme insertion occurring during expression of PpcA in E. coli are readily detected by changes in the radius of gyration, Rg, and by loss of interference features observed at wide angle that arise from the characteristic protein fold of the multi-heme cytochromes c7.14 SAXS data for each of the Ru(bpy)3-linked PpcA derivatives found that the Ru(bpy)2(Br-bpy)-labeled PpcA consisted of small globular proteins with Rg closely matching that of the wild-type protein, as well as the Rg calculated from the solution-state NMR structure.54 For example, Supporting Information, Figure S5 shows Guinier plots for the wild-type PpcA and Ru(bpy)2(Br-bpy)-labeled A23C and K29C mutants. The linear portions of the plots have slopes that provide a measure of Rg, and these are found to have closely equivalent values of 12.6 ± 0.1, 13.1 ± 0.1, and 12.8 ± 0.1 Å, respectively. Similarly, SAXS/WAXS patterns show analogous curves, Supporting Information, Figure S6, including the retention of the small interference features arising from the PpcA protein fold.14 The lack of a significant perturbation of PpcA structure upon labeling of cysteine residues with Ru(bpy)2(Br-bpy) is consistent with results and crystal structures obtained for other redox proteins labeled with this reagent.43,44 Interestingly, the Guinier plot for A23C-Ru shows a distinctive downward deviation from a linear slope that was not seen for wild-type PpcA or the K29C-Ru construct, Supporting Information, Figure S5. This type of deviation is characteristic of repulsive electrostatic inter-protein interactions,55−57 and is consistent with the net charge change upon converting a neutral alanine residue to a charge of +2 in the Ru(bpy)2(Br-bpy)-labeled mutant. Supporting Information, Figure S7 compares the SAXS/WAXS patterns for the wildtype, A23C, and A23C-Ru, and shows the appearance of the repulsive inter-protein interaction upon labeling. However, indications are not seen for increased electrostatic repulsion upon converting the lysine residue to the cysteine-linked ruthenium(II) label in the K29C-Ru complex, Figure S5. This suggests the possibility that electrostatic inter-protein interactions may depend not just on the net charge, but also the surface site location. Conformational Analysis by Molecular Dynamics Simulations. All-atom MD simulations were carried out for each of the Ru(bpy)3-linked PpcA derivatives listed in Table 1, using procedures described previously,42 and outlined above. These previous studies have shown that MD simulations D
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Figure 3. Distribution of distances of closest edge-to-edge approach between atoms forming conjugated bonds in PpcA hemes I, III, IV, with the heme numbering noted in Figure 1, and attached photosensitizer measured from conformers during MD simulations. Plots use different colors for each of three simulation trajectories for (A) K29C-Ru and (B) A23C-Ru. In the case of K29C-Ru, only the last 15 ns out of each 90−95 ns trajectory were used because of a reproducible change in label position occurring in the range of 50−75 ns in all three simulations. Mean distances and standard deviations are summarized in the Supporting Information, Table S4, and show a good agreement between data from all three simulations for each mutant.
both labeled constructs exhibit a relatively narrow, consistent distribution of distances. For both mutants, the distances of closest approach occur between the ruthenium label and heme III. For K29C-Ru the average distance of closest edge-to-edge approach is 6.8 ± 0.4 Å, and occurs between aromatic carbon atoms on the cysteine-linked pyridyl ligand and aromatic atoms on a pyrrole ring bearing a vinyl group in heme III. For A23CRu the average distance of closest edge-to-edge approach is 6.0 ± 0.8 Å, and occurs between aromatic carbon atoms on the cysteine-linked pyridyl ligand and aromatic atoms on the pyrrole ring bearing one of the propionic acids in heme III. Average distances of closest edge-to-edge approach between the ruthenium and each heme for both K29C-Ru and A23C-Ru constructs are tabulated in the Supporting Information, Table S4. Examples of representative equilibrium conformers for K29C-Ru and A23C-Ru are shown in Figures 4 and 5, respectively. In each case, the structures suggest possibilities for both close, through-space and longer, through-bond electrontransfer pathways to hemes III and I respectively. For example in K29C-Ru the thiol atom of the Cys-29 immediately precedes the two native heme ligands, Cys-30 and His-31, that form the binding site for heme I. Consequently, a short, 6-atom, through peptide bond path exists between the thiol atoms of Cys-30 covalently linked to heme and Cys-29 covalently linked to the Ru(bpy)3 photosensitizer, Figure 4A. This short through-bond pathway provides a potential path for electron transfer even though the through-space, edge-to-edge distance between Ru(bpy)3 and heme I is rather long, about 10.4 Å, as shown in Figure 3A and Supporting Information, Table S4. This linking position is also located two amino acid residues removed from His-31 which is a native axial ligand of heme I. In addition, the equilibrium conformers also show that the Ru(bpy)3 label is placed in approximate van der Waals, direct through-space contact with heme III. This is illustrated in Figure 4B, in which heme III and the Ru(bpy)3 atoms are shown plotted in their space-filling representations. A close approach is made between the methylene carbon involved in
reliably track the conformation, protein segment dynamics, and reproduce anionic porphyrin docking and PpcA multimerization detected by SAXS.42 Here, MD simulations were used to provide a further test of possible perturbation of structure and to visualize possible positioning of the linked Ru(bpy)3 with respect to the PpcA heme cofactors. Among the Ru(bpy)3-linked PpcA mutants produced above, MD simulations consistently found that thermally equilibrated conformers of the K29C-Ru and A23C-Ru constructs produced the closest through-space approach between the Ru(bpy)3 label and a heme cofactor. For example, the distances of closest edgeto-edge approach between non-hydrogen atoms involved in conjugated bonds in the photosensitizer bipyridyl ligands and each of the three hemes in the K29C-Ru and A23C-Ru constructs are shown plotted in the Supporting Information, Figures S8 and S9, respectively, and measured across the duration of the 90 ns dynamics simulations. The plots in Figures S8 and S9 show the results of three independent simulations, each initiated with different random velocities of atoms. The simulations show a consistent, relatively tight distribution of conformers. One feature seen in all three of the simulation trajectories for K29C-Ru, most noticeably in the closest atom pair distance, i.e., that between the Ru(bpy)3 label and heme IV, is a sudden jump in conformation that occurred during the final 40 ns in each simulation. This conformation jump involved a movement of the covalently attached Ru(bpy)3 and formation of stable contact with the surface of the helix comprised of residues 42−57 of PpcA. Individual conformers before and after the structural change are shown in Supporting Information, Figures S10 and S11. In all cases, the new position of Ru(bpy)3 was located between the side chains for Glu-57, Cys-54, and Lys-49 residues of PpcA. From these simulations, the distribution of distances between atoms forming conjugated bonds in PpcA hemes and the attached Ru(bpy)3 photosensitizer are shown plotted in Figure 3. Part A shows the plot for K29C-Ru, taken from the final 15 ns in all three simulations. Figure 3B shows the plot for A23CRu, taken from the full 90 ns simulations. The simulations for E
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Similarly, an example of one of the equilibrium-state conformers for A23C-Ru is shown in Figure 5A. In this case, the position of the mutation places the linked photosensitizer with a longer 15-atom peptide path between the cysteine sulfur atom linked to Ru(bpy)3 and the cysteine thiol (Cys-30) covalently linked to heme I, illustrated by the space-filling atom representation in Figure 5A. This is the shortest through-bond pathway in A23C-Ru. The addition length compared to K29CRu suggests that this would be a less favorable pathway. In addition, the equilibrium conformers for A23C-Ru also show that the Ru(bpy)3 is placed in approximate van der Waals contact with heme III, Figure 5B. Here, the distance of closest approach occurs between a bipyridyl ligand and aromatic atoms of a pyrrole ring bearing one of the propionic acid groups of heme III. This is illustrated by the space-filling atom representations for the atoms of Ru(bpy)3 and heme III, Figure 5A. The mean edge-to-edge distance between nonhydrogen aromatic atoms on the bipyridyl ligand and heme III is 6.0 Å, as noted in Table S4. This suggests that A23C-Ru could have a more favorable through-space electron transfer path compared to K29C-Ru. Correlation of Site-Directed Labeling to LightInduced Electron Transfer. Excited-state electron transfer from covalently attached Ru(bpy)3 to the PpcA heme cofactors was initiated by laser excitation using a 120 fs pulse at 460 nm, and the redox states of the hemes were followed by transient optical absorption measurements. The reaction kinetics were analyzed with the Ru(bpy)3-labeled PpcA constructs using the hemes starting the both the oxidized and reduced states, and following the reaction Schemes A and B in Figure 2. For the K29C-Ru construct starting with ferric hemes, Scheme A, we observed transient absorption difference spectra that show a clear signature for transient formation and decay of a ferrous heme charge-separated state that occurs on the picosecond time scale, Figure 4C. The time course for heme reduction and decay is plotted by the 552 nm − 541 nm difference transients, Figure 4D. 541 nm is an isosbestic point for redox changes in the heme,51 and its use as a reference wavelength allows the broad transient optical absorption changes arising from the ruthenium photosensitizer excitedand redox-state transitions to be subtracted from the heme transient kinetics. The heme absorption transients show rise time and decay of the ferrous heme charge-separated state to be 6.4 ± 0.4 and 38 ± 2 ps, respectively. Both of these processes are remarkably fast, and exceed by over 3 orders of magnitude charge-transfer rates previously reported for ruthenium-labeled single-heme proteins.27−30,32,34 Further, the transient kinetics for this ruthenium-labeled PpcA construct exceed by over a factor of 100 the fastest rates reported for rhenium photosensitizer−wire assemblies that coordinate directly to a cytochrome heme cofactor.36 We note that while the pump wavelength of 460 nm is optimal for excitation of Ru(bpy)3-based photosensitizers, a substantial fraction of pump laser pulse energy was absorbed by the three hemes of PpcA. This is illustrated by the overlap in the ground-state absorption spectrum for the K29C-Ru construct, Supporting Information, Figure S4, and by the transient absorption changes which are seen in unlabeled PpcA following 460 nm laser excitation, Supporting Information, Figure S12. The excited states of iron porphyrins are extremely short-lived,58,59 and the observed remarkably fast rate of charge separation approach the relaxation rate of the coincidentally excited ferric heme (τ = 2.66 ± 0.13 ps).
Figure 4. Representative equilibrium structure of the ferric K29C-Ru construct from molecular dynamics simulation, left, compared to lightinduced transient absorption changes for the heme cofactor. (A) Structure of the representative equilibrium conformer with six peptide atom connections between the thiol atom on Cys-29-Ru and the thiol atom on Cys-30-heme I, highlighted with the space-filling atom representation. (B) The same conformer, but with the Ru(bpy)3 photosensitizer (blue) and heme III (red) in atomic space-filling representation. (C) Transient spectra at selected time points. (D) 552 nm − 541 nm transient difference and 630 nm transient absorption kinetics.
Figure 5. Structure and charge-transfer data for oxidized A23C mutant. (A,B) Equilibrium structure of Ru(bpy)2(Br-bpy)-labeled A23C mutant of PpcA. (C) Differential spectra at selected time points, clearly showing heme reduction near 552 nm followed by charge recombination. (D) Kinetics of charge separation and recombination at 552 nm − 541 nm (black line) as well as kinetics of Ru(II)* excitedstate decay monitored at 630 nm (blue line).
the covalent bridge between Cys-29 and the bipyridyl ligand. The mean distance of closest through-space approach between non-hydrogen aromatic atoms on the bipyridyl ligand and heme III is 6.8 Å, as noted in Table S4. This represents the shortest path for edge-to-edge, through-space electron transfer in K29C-Ru. F
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Figure 6. (A) Transient spectrum averaged during the time window of 27−35 ps following 460 nm laser excitation of the dithionite-reduced K29CRu construct. The transient spectrum shows the clear signature of transient heme oxidation. (B) Kinetics of the transient absorption changes taken from selected wavelengths. The black line shows the transient 552 nm − 541 nm difference kinetics measured for the dithionite-reduced K29C-Ru construct. The red line trace shows the 552 nm − 541 nm difference recorded for dithionite-reduced wild-type PpcA. The blue line trace shows the difference kinetics (ferrous K29C-Ru) − (ferrous wild-type PpcA), both measured as the 552 nm − 541 nm difference. This double difference subtracts the transient ferrous heme response, and extracts the transient ferrous heme oxidation kinetics. Superimposed on the blue line data is a fit using a combination of an exponential rise and decay, with time constants of 5.7 ± 0.3 and 128 ± 9 ps, respectively.
solution, Figures S14 and S15, and to have an exited-state lifetime decay of about 490 ns, Figure S14. Since the residual *Ru(II) emission in the K29C-Ru construct has a lifetime distinguishable from that of free Ru(bpy)3, we anticipate that this could arise either from conformers for which the Ru(bpy)3 is not in position for rapid electron transfer, or from labeling side reactions which cause a small fraction of the Ru(bpy)3 label to react at sites beside the engineered Cys residue. In either case, the residual *Ru(II) emission decay provides an estimate of the decay rate, kd, for *Ru(II) in Ru(bpy)3-labeled PpcA constructs, and shows that the condition kCS ≫ kd is met. Photo-excited electron transfer for the K29C-Ru construct was also examined starting with the hemes in the ferrous redox state, reaction Scheme B, Figure 2. For ferrous K29C-Ru, transient absorption difference spectra show a clear signature of a charge-separated state that involved transient oxidation to a ferric heme state, Figure 6A. Similar to the experiments starting with K29C-Ru in the ferric state, the ferrous hemes show strong excitation by the 460 nm laser pulse and excited-state decay kinetics, Supporting Information, Figure S12. However, in this case, the distinction between heme excited-state decay from direct laser excitation and charge-separated-state decay from photosensitizer-initiated electron transfer was made more difficult because the longer lifetime of the ferrous heme excited state, measured to be 6.3 ± 0.1 ps, Figure S12, overlaps the time scale for electron transfer, Figure 6B. Kinetic data for ferrous K29C-Ru were corrected for the contribution from direct laser excitation of the hemes by subtracting a scaled 552 nm − 541 nm difference measured from laser excitation of ferrous wild-type PpcA. This double difference subtracts the transient ferrous heme excited-state response, and extracts the transient ferrous heme oxidation kinetics, Figure 6B. The resulting transients show a photo-initiated oxidation of the heme with a rise time and decay of 5.7 ± 0.3 and 128 ± 9 ps, respectively. Further, spectral changes approximately 30 ps after laser excitation, Figure 6A, were clearly indicative of heme oxidation and could not be assigned to the relaxation dynamics of ferrous hemes, as the contribution of the latter should be negligible at a time point corresponding to approximately 5 lifetime constants for the excited state. Similar to the results seen above for ferric K29C-Ru construct, the 630 nm *Ru(II) excited-state decay kinetics for
Figure 4D also provides a comparison of Ru(bpy)3 excitedstate decay, measured as the absorption transient at 630 nm, and kinetics of charge separation measured by the 552 nm − 541 nm difference transient. Within a three-state photochemical reaction scheme, *Ru(II) decay is expected to match the formation of the charge-separated state with a rate constant of kCS+ kd, Scheme A and eqs 1 and 2. However, in K29C-Ru, *Ru(II) is found to have multiple decay components. The data in Figure 4D were fit with 0.8 ± 0.03 and 6.4 ± 0.4 ps lifetime decay components. The 552 nm − 541 nm transient absorption rise time matches the *Ru(II) 6.4 ps decay component and is consistent with interpretation of this time constant as charge separation within the ferric K29C-Ru construct and 38 ps as the time constant for charge recombination. In addition, the K29CRu construct shows an additional 0.8 ps kinetic component which is absent in kinetic traces measured for free Ru(bpy)3 and Ru(bpy)2(Br-bpy) covalently linked to cysteine, Supporting Information, Figure S13. Energy transfer to heme is a likely mechanism for rapid *Ru(II) excited-state decay.60,61 However, the 0.8 ps decay time is remarkably short and could be suggestive of accelerated intersystem crossing for *Ru(II) in the K29C-Ru construct, for example, possibly through spin-orbit or paramagnetic coupling by heme in close contact to *Ru(II). In any mechanism, the 0.8 ps phase of the excited-state decay must be associated with conformations that differ from those undergoing 6.4 ps excited-state electron transfer, although these could involve slighteven angstrom scale or lessdifferences in the details of the *Ru(II)-heme contact. Additionally, these conformers must be interconverting on time scales comparable to, or slower than, the charge separation lifetime. On-going experiments are investigating the accelerated excited-state decay and ground-state recovery kinetics in the Ru(bpy)3 linked PpcA derivatives. We note that the *Ru(II) excited state in the K29C-Ru construct shows multi-exponential decay to the baseline in Figure 4D, fit with 0.8 and 6.4 ps biexponential time constants. The *Ru(II) excited-state decay kinetics show the lack of a clearly resolved kinetic component with a lifetime corresponding to the 590 ns emissive decay from the *Ru(II) MLCT state in solution, Supporting Information, Figures S13 and S14. Corroborating this, steady-state luminescence for K29C-Ru was found to be about 10% of that measured for Ru(bpy)3 in G
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The Journal of Physical Chemistry B the ferrous form showed multiple kinetic components all on the picosecond time scale, Supporting Information, Figure S16, of which one could be correlated with the time constant for formation of charge-separated state (5.7 ps). The remaining two time constants were 0.71 ± 0.16 and 94 ± 15 ps. These result are suggestive of both non-electron-transfer and electrontransfer quenching of the *Ru(II) excited state as described above. The comparison of the light-induced transient redox kinetics of the ferric and ferrous K29C-Ru is of interest since it demonstrates that the ruthenium-labeled constructs support bidirectional excited-state electron transfer. For K29C-Ru excited-state oxidation of a ferrous heme appears to be slightly faster, 5.7 ± 0.2 ps, than reduction of the ferric heme reduction, 6.4 ± 0.4 ps, while displaying a longer lifetime, showing charge recombination times of 128 ± 9 and 38 ± 2 ps, respectively. Analogous assays for excited-state electron transfer were also examined for the A23C-Ru construct. Starting with the ferric A23C-Ru construct, transient absorption measurements show that laser excitation of the Ru(bpy)3 photosensitizer initiates rapid heme reduction with a time constant of 130 ± 7 ps, which was followed by slower charge recombination with a time constant of 1.23 ± 0.11 ns, Figure 5C,D. While both of these processes are remarkably fast, lifetimes for the formation and decay of the charge-separated state in ferric A23C-Ru are a factor of 20- and 30-fold slower than the corresponding kinetics for electron transfer in ferric K29C-Ru. Similar to *Ru(II) excited-state data for the K29C-Ru construct, excited-state decay kinetics for ferric A23C-Ru is also multi-phasic, with a least three kinetic components at ∼0.75, 3.4, and 130 ps (Figure 5D). As with K29C-Ru, bi-directionality of the light-initiated electron transfer can also be demonstrated with A23C-Ru. Starting with dithionite reduced, ferrous A23C-Ru, excitation was found to initiate heme oxidation with a time constant of 113 ± 24 ps, and charge recombination with a time constant of 3.8 ± 0.7 ns, Supporting Information, Figure S17. As in all previously described experiments, the excited-state decay kinetics at 630 nm was multiphasic with two components at 0.9 and 7.0 ps in addition to a component similar to the time scale of the rising edge of 552 nm − 541 nm kinetics (113 ps). However, we note that in this experiment, the yield of charge separation was about a factor of 10 smaller than that seen either starting from the ferric A23C-Ru state, Figure 5D, or from the ferrous K29C-Ru construct, Figure 6B. The cause for this low yield is not known, but as discussed further below, this could arise from a conformation change for the ferrous A23C-Ru construct in this experiment. Further work is ongoing to determine the source for this low yield. Besides the A23C-Ru and K29C-Ru, we also carried out excited-state charge transfer studies on the K7C-Ru, E39C-Ru, and M45C-Ru constructs. MD simulations suggest that thermally equilibrated conformers for these constructs can be expected to have approximate distances of closest edge-to-edge approach of 10, 11, and 9 Å, respectively, as noted in Table 1. A summary of transient heme reduction kinetics for each of the five ruthenium-labeled PpcA mutants, photo-induced from the starting ferric heme state, is shown in Figure 7. The figure also shows structures for representative equilibrium conformers for each of the ruthenium-labeled constructs. The kinetic transients show charge separation and decays which span more than 4 orders of magnitude in reaction time.
Figure 7. Summary of transient absorption kinetics (552 nm − 541 nm) measured for Ru(bpy)3-ferri-PpcA constructs.
A general trend is seen between rates of electron transfer and the photosensitizer-to-heme distance. Except for the A23C-Ru and K29C-Ru constructs discussed above, the excited-state electron-transfer rates are found to qualitatively follow the sequence expected from the estimated distance between the Ru(bpy)3 photosensitizer and heme cofactor in the MD equilibrium structures. Further, the charge separation rise and decay kinetics are seen to have a similar shape, but displaced in the time in concert with a shift in the electron-transfer path. Figure 8 shows a comparison of charge separation and decay kinetics initiated from the M45C-Ru, K7C-Ru, and E39C-Ru constructs starting from the ferric and ferrous heme redox states. The plots further demonstrate the ability of the Ru(bpy)3-labeled PpcA constructs to support both oxidative and reductive quenching of the Ru(bpy)3 excited state, depending upon the initial oxidation state of the heme cofactors. In each case, and with the K29C-Ru construct discussed above, the charge separation is found to be faster starting from the ferrous, Scheme A, compared to ferric hemes, Scheme B. Exponential fits to 552 nm − 541 nm difference transients measured for the M45C-Ru, K7C-Ru, and E39C-Ru constructs are listed in the legend to Figure 8. The magnitude of the acceleration was found to vary in the different constructs. For example, the M45C-Ru, K7C-Ru, and E39C-Ru constructs show increases by factors of 2.8, 2.2, and 4.7 in the rates for charge separation starting from the ferrous compared to ferric heme states, respectively, and this compares to the factors of 1.1 and 1.2 seen for the K29C-Ru and A23C-Ru constructs, respectively. The variability in the extent of this acceleration points to the likelihood that the Ru(bpy)3-labeled constructs vary in the extents of conformational differences between the ferric and ferrous forms.
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DISCUSSION Previous work on the measurement of photo-induced electron transfer in photosensitizer-labeled proteins found that rates for electron transfer can be best correlated to pathways for electron transfer.22−26,62−65 Plots of distances between electron donor and acceptor groups have provided useful guides for generally predicting electron tunneling rates through proteins, although the scatter in these plots, corresponding to differing rates at comparable distances seen in different protein experiments, is understood to arise from the effects of specific pathways available in each protein.23−26,63−69 The results for the ruthenium-labeled PpcA cysteine mutants fit this view. However, compared to previous studies for photoH
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Figure 8. Comparison of charge separation kinetics initiated from the ferric (blue filled square traces) and ferrous (black open circle traces) heme redox states for the constructs (A) M45C-Ru, (B) K7C-Ru, and (C) E39C-Ru. Transient changes in heme redox state were measured as the 551 nm − 540 nm difference transients. The red lines are fits using the sum of an exponential rise and decay. In (A) the reductive rise and recombination decay times fit to the ferric M45C-Ru trace were (1.49 ± 0.08) × 10−8 and (5.36 ± 0.26) × 10−8 s, respectively. The oxidative rise and recombination decay times fit to the ferrous M45C-Ru trace were (5.6 ± 0.02) × 10−9 and (2.77 ± 0.08) × 10−8 s, respectively. In (B) the reductive rise and recombination decay times fit to the ferric K7C-Ru trace were (1.65 ± 0.15) × 10−8 and (1.0 ± 0.09) × 10−7 s, respectively. The oxidative rise and recombination decay times fit to the ferrous K7C-Ru trace were (7.53 ± 0.38) × 10−9 and (1.42 ± 0.08) × 10−7 s, respectively. In (C), the reductive rise and recombination decay times fit to the ferric E39C-Ru trace were (3.53 ± 0.09) × 10−8 and (2.61 ± 0.08) × 10−7 s, respectively. The oxidative rise and recombination decay times fit to the ferrous E39C-Ru trace were (7.52 ± 0.38) × 10−9 and (1.12 ± 0.05) × 10−7 s, respectively.
Decay kinetics of *Ru(II) excited state for both K29C-Ru and A23C-Ru constructs showed multiple components indicative of both electron-transfer (ET) and non-electrontransfer (non-ET) quenching of the *Ru(II) by the heme cofactors in the PpcA-Ru constructs. A remarkable feature of the non-ET quenching is the fast 0.8 ps phase of the excitedstate decay which must be associated with conformations that differ from those undergoing 6.4 ps excited-state ET. This suggests that rapid non-ET quenching could be operating through short-range mechanisms like direct orbital overlap that fall off more rapidly with distance than the through-bond pathways used for ET. From this view, the ultrafast non-ET quenching could be expected to be more sensitive to details of the atom pair distance between Ru(bpy)3 and heme than ET quenching. The MD simulations show that the Ru(bpy)3 and heme atom pair distances are spread across a distribution, and that jumps between conformer states occur on the nanosecond time scale. This would suggest that the experimental excitedstate decay measurements on the picosecond time scale provide a snapshot of the conformational distribution. The photo-induced ET rates for the Ru(bpy)3 linked PpcA constructs can be considered in the context of the Marcus theory for ET. The reduction potential Fe(III/II) for the three PpcA heme is centered at about −0.15 V (vs NHE),13,73 and the excited-state redox potentials for Ru(II*/III)(bpy)3 and Ru(II*/I)(bpy)3 are at about −0.83 and −0.79 V, respectively.49,74,75 These values yield free energies for PpcA photooxidation and photo-reduction of about −0.94 and −0.68 V, respectively. From this and the reaction Schemes A and B in Figure 2, free energies for charge recombination from the Fe(II)−Ru(III) charge-separated state can be estimated to be about −1.32 eV, Scheme A, while charge recombination from the Fe(III)−Ru(II)L− charge-separated state would have a driving force of about −1.06 eV, Scheme B. A summary of rates and driving force for each of the four ET steps in reactions Schemes A and B for each of the five PpcA-Ru constructs is given in the Supporting Information, Table S5. The relationship between driving force and ET rate for the five PpcA-Ru constructs is also shown plotted in Figure 9. The data for K29C-Ru and A23C-Ru are found to follow Marcus behavior,76,77 using a reorganization energy of 0.85 V and scaling the electronic coupling pre-exponential factor to fit the
induced electron transfer in photosensitizer-labeled redox proteins, the A23C-Ru and K29C-Ru constructs are of interest because they are in position to make direct contacts to heme III. The MD simulations of labeled A23C and K29C were in good agreement with X-ray scattering measurements and the simulations revealed relatively narrow distributions of the shortest distances between atoms involved in conjugated bonds of the hemes and photosensitizer. The MD simulations predicted an approximately 0.8 Å shorter distance between heme III and the photosensitizer label in ferric A23C-Ru compared to ferric K29C-Ru, although 20-fold faster charge separation and decay rates are seen for ferric K29C-Ru compared to A23C-Ru. The structures suggest that the faster rates in K29C-Ru compared to A23C-Ru can be understood because of the shorter through-peptide pathway to heme I for K29C-Ru compared to A23C-Ru, and by the through-space pathways to heme III which involve the conjugated vinyl group in K29C-Ru compared to the saturated atom paths through the heme III propionic acid group for A23C-Ru. Both A23C-Ru and K29C-Ru constructs are noteworthy because of the ultrafast excited-state electron transfer. In particular, the charge separation risetimes of 6.4 ps for heme photo-reduction and 5.7 ps for heme photo-oxidation in ferric and ferrous K29C-Ru, respectively, are exceptional and nearly 4 orders of magnitude faster than the fastest rates reported for electron-transfer redox proteins with photosensitizers linked at amino acid sites,23,31,32 and more than a factor of 200-fold faster than electron-transfer rates using photosensitizer wires directly linked to the heme cofactor.37,70−72 From the comparison of photo-induced charge separation and decay across the series of the ruthenium-labeled PpcA mutants examined in this study, one feature that stands out is the consistency of the kinetic response. In all cases, the chargeseparated-state rise and decay kinetics have similar shapes, but essentially shifted in time corresponding to the shift in the site for Ru(bpy)3 attachment. There appears to be no clear extension of the charge-separated-state lifetime upon injecting the electron or hole into the multi-heme array. Electron exchange between PpcA heme sites has been shown to be rapid on an NMR time scale,11−13 corresponding to exchange times shorter than 10−5 s. I
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multi-heme cytochrome frameworks to investigate electron transfer in protein “molecular wires” and to serve as frameworks for metalloprotein designs that support multiple-electrontransfer redox chemistry.
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ASSOCIATED CONTENT
S Supporting Information *
List of the oligonucleotide primers used for PCR. List of PDB entries for c-type cytochromes with at least one addition cysteine residue not used in heme linking. Sequence alignment of G. sulfureducens PpcA with closely related proteins. Table summarizing the averaged closest edge-to-edge distances between the Ru(bpy)3 label and hemes in the A23C-Ru and K29C-Ru constructs extracted from MD trajectories. Table summarizing photoinduced electron-transfer lifetimes, reaction driving force, and closest edge-to-edge distance between Ru(bpy)3 and heme groups for five Ru(bpy)3-linked mutant PpcA constructs. ESI-MS data showing reversible binding of an unidentified E. coli metabolite to free Cys residues of PpcA. Representative LC elution profiles for PpcA and Ru(Bpy)3labeled PpcA mutants. ESI-MS data for Ru(Bpy)3-labeled PpcA mutants. Comparison of optical absorption for Ru(bpy)2(bpyCH2Br), K29C, and the K29C-Ru construct. Guinier plots from SAXS. SAXS/WAXS scattering patterns for PpcA, K29C-Ru, and A23C-Ru constructs. Plots of the closest edge-to-edge distances between Ru(Bpy)3 and heme in the K29C-Ru and A23C-Ru constructs from MD simulations. Representative examples of conformer structures taken from MD simulation trajectories. Heme ultrafast transient absorption changes induced by 460 nm laser excitation. Comparison of excitedstate Ru* kinetics for Ru(bpy)3 and Ru(bpy)2(bpy-Cys). Luminescence emission decay times for K29C-Ru and Ru(bpy)3Cl2 by time-correlated single-photon counting. Luminescence spectra for Ru(bpy)3Cl2 and K29C-Ru, measured using 460 nm excitation. Ultrafast transient absorbance kinetics of *Ru(II) decay in ferrous K29C-Ru measured by the absorption transient at 630 nm. Transient absorption kinetics measured for the dithionite-reduced, ferrous A23C-Ru construct. This material is available free of charge via the Internet at http://pubs.acs.org.
Figure 9. Relationship between driving force, ΔG0, and electrontransfer (ET) rate for the five PpcA-Ru constructs. The ET rates correspond to the charge separation and recombination reactions labeled A-CS, A-CR from the reactions in Schemes A, and B-CS, B-CR for the charge separation and recombination reactions in Scheme B. Data are marked by symbols, corresponding to K29C-Ru (black squares), A23C-Ru (red circles), M45C-Ru (blue triangles), K7C-Ru (magenta inverted triangles), and E39C-Ru (green diamonds). The lines drawn through the K29C-Ru and A23C-Ru data were calculated from the Marcus ET equation: kET = EC×(4πλkbT)−1/2 exp[−(λ + ΔGo)2/4λkbT], using λ = 0.85 V, T = 298 K, and the electronic coupling term, EC, scaled to fit the data, with a value of 1.12 × 1011 s−1 for K29C-Ru and 5.3 × 109 s−1 for A23C-Ru.
experimental data. It is interesting to note that the charge recombination reaction from Fe(III)−Ru(II)L−, labeled B-CR in Figure 9, is an outlier point for both K29C-Ru and A23C-Ru, and to a lesser extent for the other three labeling sites as well. This implies that charge recombination from Fe(III)−Ru(II)L− occurs from a configuration with ET parameters that differ from the other reactions, and that the configuration change occurs following the initial charge separation. NMR structures have demonstrated structural differences between the ferric and ferrous forms in the homologous PpcC,20 and oxidation of the PpcA hemes is known to cause pKa shifts and Bohr proton function.11,45,78 The MD simulations for the PpcA-Ru constructs have been carried out with the hemes in the ferric states. These simulations resolve conformational changes, seen by the jumps in the Ru(bpy)3-to-heme distances, Supporting Information, Figures S8 and S9, that occur on the nanosecond time scale and are too slow to occur during the lifetime of the Fe(III)−Ru(II)L− charge-separated state. This suggests the speculation that the configuration change could involve local structural change, for example proton transfers that serve as the trigger for larger conformation change and Bohr proton release. Further experiments and dynamics simulations involving Ru(bpy)3 and heme oxidation-state changes are ongoing to test these concepts. Finally, we note that bidirectionality plus the capability for picosecond-time-scale electron transfer to tethered photosensitizers imparts an ability to inject either electrons or holes on the picosecond scale within the multi-heme cytrochrome. This is significant since it creates opportunities to exploit chromophores with short excited-state lifetime, including photosensitizers based on abundant transition metals, and to link multi-heme cytochrome redox chemistry to the short-lived charge-separated states in photosensitizer−catalyst dyads.48,79,80 This work demonstrate an opportunity to develop
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Present Address #
O.K.: Department of Chemistry and Biochemistry, James Madison University, 901 Carrier Drive, Harrisonburg, VA 22807 Author Contributions §
N.S.P. is a consultant contracted by the Argonne National Laboratory from Lab Support, 9450 Bryn Mawr Ave., #340, Rosemont, IL 60018. All work was performed at the Argonne National Laboratory. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy under Contract DE-AC0206CH11357. X-ray scattering experiments were carried out at beamline 12-ID-B of the Advanced Photon Source, an Office of J
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Center Shows Unexpected Changes in Main Chain Orientations and Quinone Position. Biochemistry 2002, 41, 5998−6007. (16) Pokkuluri, P. R.; Londer, Y. Y.; Duke, N. E. C.; Erickson, J.; Pessanha, M.; Salgueiro, C. A.; Schiffer, M. Structure of a Novel c7Type Three-Heme Cytochrome Domain from a Multidomain Cytochrome c Polymer. Protein Sci. 2004, 13, 1684−1692. (17) Pokkuluri, P. R.; Londer, Y. Y.; Duke, N. E. C.; Long, W. C.; Schiffer, M. Family of Cytochrome c7-Type Proteins from Geobacter Sulfurreducens: Structure of One Cytochrome c7 at 1.45 Angstrom Resolution. Biochemistry 2004, 43, 849−859. (18) Londer, Y. Y.; Pokkuluri, P. R.; Erickson, J.; Orshonsky, Y.; Schiffer, M. Heterologous Expression of Hexaheme Fragments of a Multidomain Cytochrome from Geobacter Sulfurreducens Representing a Novel Class of Cytochromes c. Protein Exp. Purif. 2005, 39, 254− 260. (19) Londer, Y. Y.; Pokkuluri, P. R.; Orshonsky, V.; Orshonsky, L.; Schiffer, M. Heterologous Expression of Dodecaheme “Nanowire” Cytochromes c from Geobacter Sulfurreducens. Protein Exp. Purif. 2006, 47, 241−248. (20) Morgado, L.; Bruix, M.; Londer, Y. Y.; Pokkuluri, P. R.; Schiffer, M.; Salgueiro, C. A. Redox-Linked Conformational Changes of a Multiheme Cytochrome from Geobacter Sulfurreducens. Biochem. Biophys. Res. Commun. 2007, 360, 194−198. (21) Pokkuluri, P. R.; Pessanha, M.; Londer, Y. Y.; Wood, S. J.; Duke, N. E. C.; Wilton, R.; Catarino, T.; Saigueiro, C. A.; Schiffer, M. Structures and Solution Properties of Two Novel Periplasmic Sensor Domains with c-Type Heme from Chemotaxis Proteins of Geobacter Sulfurreducens: Implications for Signal Transduction. J. Mol. Biol. 2008, 377, 1498−1517. (22) Winkler, J. R.; Gray, H. B. Electron Transfer in RutheniumModified Proteins. Chem. Rev. 1992, 92, 369−379. (23) Winkler, J. R.; Gray, H. B. Electron Flow through Metalloproteins. Chem. Rev. 2014, 114, 3369−3380. (24) Winkler, J. R.; Gray, H. B. Long-Range Electron Tunneling. J. Am. Chem. Soc. 2014, 136, 2930−2939. (25) Gray, H. B.; Winkler, J. R. Electron Flow through Proteins. Chem. Phys. Lett. 2009, 483, 1−9. (26) Warren, J. J.; Ener, M. E.; Vlcek, A.; Winkler, J. R.; Gray, H. B. Electron Hopping through Proteins. Coord. Chem. Rev. 2012, 256, 2478−2487. (27) Wuttke, D. S.; Bjerrum, M. J.; Winkler, J. R.; Gray, H. B. Electron-Tunneling Pathways in Cytochrome C. Science 1992, 256, 1007−1009. (28) Chang, I.-J.; Gray, H. B.; Winkler, J. R. High-Driving-Force Electron Transfer in Metalloproteins: Intramolecular Oxidation of Ferrocytochrome c by Ru(2,2′-Bpy)2(Im)(His-33)3+. J. Am. Chem. Soc. 1991, 113, 7056−7057. (29) Mines, G. A.; Bjerrum, M. J.; Hill, M. G.; Casimiro, D. R.; Chang, I.-J.; Winkler, J. R.; Gray, H. B. Rates of Heme Oxidation and Reduction in Ru(His33)Cytochrome C at Very High Driving Forces. J. Am. Chem. Soc. 1989, 118, 1961−1965. (30) Wuttke, D. S.; Gray, H. B.; Fisher, S. L.; Imperiali, B. Semisynthesis of Bipyridyl-Alanine Cytochrome c Mutants: Novel Proteins with Enhanced Electron-Transfer Properties. J. Am. Chem. Soc. 1993, 115, 8455−8456. (31) Pan, L. P.; Durham, B.; Wolinska, J.; Millett, F. Preparation and Characterization of Singly Labeled Ruthenium Polypyridine Cytochrome C Derivatives. Biochemistry 1988, 27, 7180−7184. (32) Durham, B.; Pan, L. P.; Long, J. E.; Millett, F. Photoinduced Electron-Transfer Kinetics of Singly Labeled Ruthenium Bis(Bipyridine) Dicarboxybipyridine Cytochrome c Derivatives. Biochemistry 1989, 28, 8659−8665. (33) Geren, L.; Hahm, S.; Durham, B.; Millett, F. Photoinduced Electron Transfer between Cytochrome C Peroxidase and Yeast Cytochrome c Labeled at Cys 102 with (4-Bromomethyl4′methylbipyridine)[Bis(Bipyridine)]Ruthenium2+. Biochemistry 1991, 30, 9450−9457. (34) Durham, B.; Millett, F. Design of Photoactive Ruthenium Complexes to Study Electron Transfer and Proton Pumping in
Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science supported at Argonne National Laboratory by the U.S. DOE under Contract No. DE-AC02-06CH11357. The authors gratefully acknowledge help of Dr. Xiaobing Zuo and staff of Sector 12 of Advanced Photon Source. We also gratefully acknowledge the computing resources provided on ″Fusion,″ a 320-node computing cluster operated by the Laboratory Computing Resource Center at Argonne National Laboratory for the molecular dynamics simulations. Use of the Transient Absorption Facility at the Center for Nanoscale Materials was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357.
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REFERENCES
(1) Bewley, K. D.; Ellis, K. E.; Firer-Sherwood, M. A.; Elliott, S. J. Multi-Heme Proteins: Nature’s Electronic Multi-Purpose Tool. Biochim. Biophys. Acta-Bioenerg 2013, 1827, 938−948. (2) Paquete, C. M.; Louro, R. O. Unveiling the Details of Electron Transfer in Multicenter Redox Proteins. Acc. Chem. Res. 2014, 47, 56− 65. (3) Breuer, M.; Rosso, K. M.; Blumberger, J. Electron Flow in Multiheme Bacterial Cytochromes Is a Balancing Act between Heme Electronic Interaction and Redox Potentials. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 611−616. (4) Bonanni, P. S.; Massazza, D.; Busalmen, J. P. Stepping Stones in the Electron Transport from Cells to Electrodes in Geobacter Sulfurreducens Biofilms. Phys. Chem. Chem. Phys. 2013, 15, 10300− 10306. (5) Shi, L.; Squier, T. C.; Zachara, J. M.; Fredrickson, J. K. Respiration of Metal (Hydr)Oxides by Shewanella and Geobacter: A Key Role for Multihaem c-Type Cytochromes. Mol. Microbiol. 2007, 65, 12−20. (6) Mowat, C. G.; Chapman, S. K. Multi-Heme Cytochromes - New Structures, New Chemistry. Dalton Trans. 2005, 21, 3381−3389. (7) Louro, R. O. Proton Thrusters: Overview of the Structural and Functional Features of Soluble Tetrahaem Cytochromes c3. J. Biol. Inorg. Chem. 2007, 12, 1−10. (8) Akutsu, H.; Takayama, Y. Functional Roles of the Heme Architecture and Its Environment in Tetraheme Cytochrome c. Acc. Chem. Res. 2007, 40, 171−178. (9) Firer-Sherwood, M.; Pulcu, G. S.; Elliott, S. J. Electrochemical Interrogations of the Mtr Cytochromes from Shewanella: Opening a Potential Window. J. Biol. Inorg. Chem. 2008, 13, 849−854. (10) Lovley, D. R. Extracellular Electron Transfer: Wires, Capacitors, Iron Lungs, and More. Geobiology 2008, 6, 225−231. (11) Morgado, L.; Bruix, M.; Orshonsky, V.; Londer, Y. Y.; Duke, N. E. C.; Yang, X. J.; Pokkuluri, P. R.; Schiffer, M.; Salgueiro, C. A. Structural Insights into the Modulation of the Redox Properties of Two Geobacter Sulfurreducens Homologous Triheme Cytochromes. Biochim. Biophys. Acta-Bioenerg. 2008, 1777, 1157−1165. (12) Pessanha, M.; Morgado, L.; Louro, R. O.; Londer, Y. Y.; Pokkuluri, P. R.; Schiffer, M.; Salgueiro, C. A. Thermodynamic Characterization of Triheme Cytochrome Ppca from Geobacter Sulfurreducens: Evidence for a Role Played in E(−)/H+ Energy Transduction. Biochemistry 2006, 45, 13910−13917. (13) Pessanha, M.; Londer, Y. Y.; Long, W. C.; Erickson, J.; Pokkuluri, P. R.; Schiffer, M.; Salgueiro, C. A. Redox Characterization of Geobacter Sulfurreducens Cytochrome c7: Physiological Relevance of the Conserved Residue F15 Probed by Site-Specific Mutagenesis. Biochemistry 2004, 43, 9909−9917. (14) Londer, Y. Y.; Pokkuluri, P.; Tiede, D. M.; Schiffer, M. Production and Preliminary Characterization of a Recombinant Triheme Cytochrome c7 from Geobacter Sulfurreducens in Escherichia Coli. Biochim. Biophys. Acta-Bioenerg. 2002, 1554, 202−211. (15) Pokkuluri, P.; Laible, P. D.; Deng, Y.-L.; Wong, T. N.; Hanson, D. K.; Schiffer, M. The Structure of a Mutant Photosynthetic Reaction K
DOI: 10.1021/jp511558f J. Phys. Chem. B XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry B Cytochrome Oxidase. Biochim. Biophys. Acta-Bioenerg. 2012, 1817, 567−574. (35) Hvasanov, D.; Mason, A. F.; Goldstein, D. C.; Bhadbhade, M.; Thordarson, P. Optimising the Synthesis, Polymer Membrane Encapsulation and Photoreduction Performance of Ru(II)- and Ir(III)-Bis(Terpyridine) Cytochrome c Bioconjugates. Org. Biomol. Chem. 2013, 11, 4602−4612. (36) Belliston-Bittner, W.; Dunn, A. R.; Nguyen, Y. H. L.; Stuehr, D. J.; Winkler, J. R.; Gray, H. B. Picosecond Photoreduction of Inducible Nitric Oxide Synthase by Rhenium(I)-Diimine Wires. J. Am. Chem. Soc. 2005, 127, 15907−15915. (37) Hartings, M. R.; Kurnikov, I. V.; Dunn, A. R.; Winkler, J. R.; Gray, H. B.; Ratner, M. A. Electron Tunneling through Sensitizer Wires Bound to Proteins. Coord. Chem. Rev. 2010, 254, 248−253. (38) Jankowska, K. I.; Pagba, C. V.; Piatnitski Chekler, E. L.; Deshayes, K.; Piotrowiak, P. Electrostatic Docking of a Supramolecular Host-Guest Assembly to Cytochrome c Probed by Bidirectional Photoinduced Electron Transfer. J. Am. Chem. Soc. 2010, 132, 16423− 16431. (39) Co, N. P.; Young, R. M.; Smeigh, A. L.; Wasielewski, M. R.; Hoffman, B. M. Symmetrized Photoinitiated Electron Flow within the Myoglobin:Cytochrome b5 Complex on Singlet and Triplet Time Scales: Energetics Vs Dynamics. J. Am. Chem. Soc. 2014, 136, 12730− 12736. (40) Xiong, P.; Nocek, J. M.; Vura-Weis, J.; Lockard, J. V.; Wasielewski, M. R.; Hoffman, B. M. Faster Interprotein Electron Transfer in a [Myoglobin, b5] Complex with a Redesigned Interface. Science 2010, 330, 1075−1078. (41) Sun, L.; Berglund, H.; Davydov, R.; Norrby, T.; Hammarström, L.; Korall, P.; Börje, A.; Philouze, C.; Berg, K.; Tran, A.; Andersson, M.; Stenhagen, G.; Mårtensson, J.; Almgren, M.; Styring, S.; Åkermark, B. Binuclear Ruthenium−Manganese Complexes as Simple Artificial Models for Photosystem II in Green Plants. J. Am. Chem. Soc. 1997, 119, 6996−7004. (42) Kokhan, O.; Ponomarenko, N.; Pokkuluri, P. R.; Schiffer, M.; Tiede, D. M. Multimerization of Solution-State Proteins by Tetrakis(4Sulfonatophenyl)Porphyrin. Biochemistry 2014, 53, 5070−5079. (43) Halavaty, A.; Müller, J. J.; Contzen, J.; Jung, C.; Hannemann, F.; Bernhardt, R.; Galander, M.; Lendzian, F.; Heinemann, U. LightInduced Reduction of Bovine Adrenodoxin Via the Covalently Bound Ruthenium(Ii) Bipyridyl Complex: Intramolecular Electron Transfer and Crystal Structure. Biochemistry 2005, 45, 709−718. (44) Contzen, J.; Kostka, S.; Kraft, R.; Jung, C. Intermolecular Electron Transfer in Cytochrome P450cam Covalently Bound with Tris(2,2′-Bipyridyl)Ruthenium(Ii): Structural Changes Detected by FTIR Spectroscopy. J. Inorg. Biochem. 2002, 91, 607−617. (45) Morgado, L.; Paixão, V. B.; Schiffer, M.; Pokkuluri, P. R.; Bruix, M.; Salgueiro, C. A. Revealing the Structural Origin of the Redox-Bohr Effect: The First Solution Structure of a Cytochrome from Geobacter Sulfurreducens. Biochem. J. 2012, 441, 179−187. (46) Humphrey, W.; Dalke, A.; Schulten, K. Vmd: Visual Molecular Dynamics. J. Mol. Graphics 1996, 14, 33−38. (47) Phillips, J. C.; Braun, R.; Wang, W.; Gumbart, J. C.; Tajkhorshid, E.; Villa, E.; Chipot, C.; Skeel, R. D.; Kale, L.; Schulten, K. Scalable Molecular Dynamics with Namd. J. Comput. Chem. 2005, 26, 1781− 1802. (48) Mukherjee, A.; Kokhan, O.; Huang, J.; Niklas, J.; Chen, L. X.; Tiede, D. M.; Mulfort, K. L. Detection of a Charge-Separated Catalyst Precursor State in a Linked Photosensitizer-Catalyst Assembly. Phys. Chem. Chem. Phys. 2013, 15, 21070−21076. (49) Kalyanasundaram, K.; Gratzel, M. Photosensitization and Photocatalysis Using Inorganic and Organometallic Compounds. In Catalysis by Metal Complexes; Ugo, R., James, B. R., Eds.; Kluwer Academic Publishers: Amsterdam, 1993; Vol. 14. (50) Yeh, A. T.; Shank, C. V.; McCusker, J. K. Ultrafast Electron Localization Dynamics Following Photo-Induced Charge Transfer. Science 2000, 289, 935−938. (51) Meyer, T. E.; Kamen, M. D. New Perspectives on c-Type Cytochromes. Adv. Protein Chem. 1982, 35, 105−212.
(52) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. The Protein Data Bank. Nucleic Acids Res. 2000, 28, 235−242. (53) Allen, J. W. A.; Sawyer, E. B.; Ginger, M. L.; Barker, P. D.; Ferguson, S. J. Variant C-Type Cytochromes as Probes of the Substrate Specificity of the E. Coli Cytochrome c Maturation (Ccm) Apparatus. Biochem. J. 2009, 419, 177−184. (54) Dantas, J. M.; Morgado, L.; Pokkuluri, P. R.; Turner, D. L.; Salgueiro, C. A. Solution Structure of a Mutant of the Triheme Cytochrome Ppca from Geobacter Sulfurreducens Sheds Light on the Role of the Conserved Aromatic Residue F15. Biochim. Biophys. ActaBioenerg. 2013, 1827, 484−492. (55) Ducruix, A.; Guilloteau, J. P.; Ries-Kautt, M.; Tardieu, A. Protein Interactions as Seen by Solution X-Ray Scattering Prior to Crystallogenesis. J. Cryst. Growth 1996, 168, 28−39. (56) Chen, S. H.; Bendedouch, D. Structure and Interactions of Proteins in Solution Studied by Small-Angle Neutron Scattering. Methods Enzymol. 1986, 130, 79−116. (57) Tiede, D. M.; Zhang, R.; Seifert, S. Protein Conformations Explored by Difference High-Angle Solution X-Ray Scattering: Oxidation State and Temperature Dependent Changes in Cytochrome c. Biochemistry 2002, 41, 6605−6614. (58) Negrerie, M.; Cianetti, S.; Vos, M. H.; Martin, J. L.; Kruglik, S. G. Ultrafast Heme Dynamics in Ferrous Versus Ferric Cytochrome C Studied by Time-Resolved Resonance Raman and Transient Absorption Spectroscopy. J. Phys. Chem. B 2006, 110, 12766−12781. (59) Bram, O.; Consani, C.; Cannizzo, A.; Chergui, M. Femtosecond Uv Studies of the Electronic Relaxation Processes in Cytochrome C. J. Phys. Chem. B 2011, 115, 13723−13730. (60) Tobin, P. H.; Wilson, C. J. Examining Photoinduced Energy Transfer in Pseudomonas Aeruginosa Azurin. J. Am. Chem. Soc. 2014, 136, 1793−1802. (61) Soler, M.; McCusker, J. K. Distinguishing between Dexter and Rapid Sequential Electron Transfer in Covalently Linked DonorAcceptor Assemblies. J. Am. Chem. Soc. 2008, 130, 4708−4724. (62) Beratan, D. N.; Liu, C. C.; Migliore, A.; Polizzi, N. F.; Skourtis, S. S.; Zhang, P.; Zhang, Y. F. Charge Transfer in Dynamical Biosystems, or the Treachery of (Static) Images. Acc. Chem. Res. 2015, 48, 474−481. (63) Skourtis, S. S.; Waldeck, D. H.; Beratan, D. N. Annu. Rev. Phys. Chem. 2010, 61, 461−485. (64) Beratan, D. N.; Onuchic, J. N.; Winkler, J. R.; Gray, H. B. Electron-Tunneling Pathways in Proteins. Science 1992, 258, 1740− 1741. (65) Beratan, D. N.; Betts, J. N.; Onuchic, J. N. Protein Electron Transfer Rates Set by the Bridging Secondary and Tertiary Structure. Science 1991, 252, 1285−1288. (66) Moser, C. C.; Keske, J. M.; Warncke, K.; Farid, R. S.; Dutton, P. L. The Nature of Biological Electron Transfer. Nature 1992, 355, 796−802. (67) Robertson, D. E.; Farid, R. S.; Moser, C. C.; Urbauer, J. L.; Mulholland, S. E.; Pidikiti, R.; Lear, J. D.; Wand, A. J.; Degrado, W. F.; Dutton, P. L. Design and Synthesis of Multi-Heme Proteins. Nature 1994, 368, 425−431. (68) Page, C. C.; Moser, C. C.; Chen, X. X.; Dutton, P. L. Natural Engineering Principles of Electron Tunnelling in Biological OxidationReduction. Nature 1999, 402, 47−52. (69) Moser, C. C.; Chobot, S. E.; Page, C. C.; Dutton, P. L. Distance Metrics for Heme Protein Electron Tunneling. Biochim. Biophys. ActaBioenerg. 2008, 1777, 1032−1037. (70) Berglund, J.; Pascher, T.; Winkler, J. R.; Gray, H. B. Photoinduced Oxidation of Horseradish Peroxidase. J. Am. Chem. Soc. 1997, 119, 2464−2469. (71) Dunn, A. R.; Dmochowski, I. J.; Winkler, J. R.; Gray, H. B. Nanosecond Photoreduction of Cytochrome P450cam by ChannelSpecific Ru-Diimine Electron Tunneling Wires. J. Am. Chem. Soc. 2003, 125, 12450−12456. L
DOI: 10.1021/jp511558f J. Phys. Chem. B XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry B (72) Warren, J. J.; Herrera, N.; Hill, M. G.; Winkler, J. R.; Gray, H. B. Electron Flow through Nitrotyrosinate in Pseudomonas Aeruginosa Azurin. J. Am. Chem. Soc. 2013, 135, 11151−11158. (73) Morgado, L.; Bruix, M.; Pessanha, M.; Londer, Y. Y.; Salgueiro, C. A. Thermodynamic Characterization of a Triheme Cytochrome Family from Geobacter Sulfurreducens Reveals Mechanistic and Functional Diversity. Biophys. J. 2010, 99, 293−301. (74) Engstrom, G.; Rajagukguk, R.; Saunders, A. J.; Patel, C. N.; Rajagukguk, S.; Merbitz-Zahradnik, T.; Xiao, K.; Pielak, G. J.; Trumpower, B.; Yu, C.-A.; Yu, L.; Durham, B.; Millett, F. Design of a Ruthenium-Labeled Cytochrome c Derivative to Study Electron Transfer with the Cytochrome Bc1 Complex. Biochemistry 2003, 42, 2816−2824. (75) Rillema, D. P.; Allen, G.; Meyer, T. J.; Conrad, D. Redox Properties of Ruthenium(II) tris Chelate Complexes Containing the Ligands 2,2′-Bipyrazine, 2,2′-Bipyridine, and 2,2′-Bipyrimidine. Inorg. Chem. 1983, 22, 1617−1622. (76) Marcus, R. A.; Sutin, N. Electron Transfers in Chemistry and Biology. Biochim. Biophys. Acta-Bioenerg. 1985, 811, 265−322. (77) Marcus, R. A. Electron-Transfer Reactions in Chemistry Theory and Experiment (Nobel Lecture). Angew. Chem., Int. Ed. 1993, 32, 1111−1121. (78) Dantas, J. M.; Morgado, L.; Catarino, T.; Kokhan, O.; Pokkuluri, P. R.; Salgueiro, C. A. Evidence for Interaction between the Triheme Cytochrome PpcA from Geobacter Sulfurreducens and Anthrahydroquinone-2,6-Disulfonate, an Analog of the Redox Active Components of Humic Substances. Biochim. Biophys. Acta-Bioenerg. 2014, 1837, 750−760. (79) Mulfort, K. L.; Tiede, D. M. Supramolecular Cobaloxime Assemblies for H2 Photocatalysis: An Initial Solution State StructureFunction Analysis. J. Phys. Chem. B 2010, 114, 14572−14581. (80) Veldkamp, B. S.; Han, W.-S.; Dyar, S. M.; Eaton, S. W.; Ratner, M. A.; Wasielewski, M. R. Photoinitiated Multi-Step Charge Separation and Ultrafast Charge Transfer Induced Dissociation in a Pyridyl-Linked Photosensitizer-Cobaloxime Assembly. Energy & Environ. Sci. 2013, 6, 1917−1928.
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DOI: 10.1021/jp511558f J. Phys. Chem. B XXXX, XXX, XXX−XXX