Multielectron Chemistry within a Model Nickel Metalloprotein

Article pubs.acs.org/JACS

Multielectron Chemistry within a Model Nickel Metalloprotein: Mechanistic Implications for Acetyl-CoA Synthase Anastasia C. Manesis, Matthew J. O’Connor,† Camille R. Schneider, and Hannah S. Shafaat* The Ohio State University, 100 West 18th Avenue, Newman & Wolfrom Laboratory of Chemistry, Columbus, Ohio 43210, United States S Supporting Information *

ABSTRACT: The acetyl coenzyme A synthase (ACS) enzyme plays a central role in the metabolism of anaerobic bacteria and archaea, catalyzing the reversible synthesis of acetyl-CoA from CO and a methyl group through a series of nickel-based organometallic intermediates. Owing to the extreme complexity of the native enzyme systems, the mechanism by which this catalysis occurs remains poorly understood. In this work, we have developed a protein-based model for the NiP center of acetyl coenzyme A synthase using a nickel-substituted azurin protein (NiAz). NiAz is the first model nickel protein system capable of accessing three (NiI/NiII/NiIII) distinct oxidation states within a physiological potential range in aqueous solution, a critical feature for achieving organometallic ACS activity, and binds CO and −CH3 groups with biologically relevant affinity. Characterization of the NiI−CO species through spectroscopic and computational techniques reveals fundamentally similar features between the model NiAz system and the native ACS enzyme, highlighting the potential for related reactivity in this model protein. This work provides insight into the enzymatic process, with implications toward engineering biological catalysts for organometallic processes.

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INTRODUCTION Nickel-containing enzymes play a central role as nature’s organometallic catalysts. The generation of direct metal− carbon bonds is found in five of the nine known nickel enzymes, and most of these systems are critically involved in the regulation of global carbon, nitrogen, hydrogen, and oxygen gas cycles (Figure 1). From detoxifying reactive oxygen species in the highly abundant Ni superoxide dismutase (Ni-SOD)1 to the utilization of atmospheric carbon dioxide by carbon monoxide dehydrogenase (CODH),2 biological Ni is found in three distinct oxidation states and can access potentials spanning over a 1.4 V range.3−5 This versatility explains the myriad of chemical transformations this first-row transition metal can achieve. Moreover, nature’s selection of nickel for reversible, two-electron redox processes involving primordial gases such as H2, CO2, and CO alludes to its likely role behind early chemoautotrophic life.6 Among all the remarkable nickel enzymes, one system of particular interest is the CODH/acetyl coenzyme A synthase (ACS) (CODH/ACS) complex. This enzyme performs both carbon dioxide fixation and organometallic carbon−carbon © 2017 American Chemical Society

bond coupling reactions within a single protein complex. Reversible reduction of CO2 to CO at the Ni center in CODH is followed by the intramolecular transport of CO through a 70 Å long hydrophobic tunnel to the nickel-containing ACS active site.7 Upon arrival at the so-called proximal nickel center, NiP, CO is combined with a methyl group and CoA to form the important metabolite and cellular building block, acetyl-CoA.5 In the absence of CoA, slow hydrolysis occurs to give acetate.8−10 While CO is known to strongly inhibit a number of enzyme systems and model compounds because of its high affinity for electron-rich systems and ability to accept metal delectrons through π-back-bonding interactions, it is an important component in the ACS system, forming a reactive metal−carbonyl species that undergoes a reversible migratory insertion reaction upon methyl addition.6 The enzymatic process thus mirrors the reaction performed in the commercially valuable Monsanto and Cativa processes for acetic acid synthesis, which require expensive rhodium or Received: April 17, 2017 Published: July 5, 2017 10328

DOI: 10.1021/jacs.7b03892 J. Am. Chem. Soc. 2017, 139, 10328−10338

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Journal of the American Chemical Society

Figure 1. Select nickel-containing enzyme systems involved in the regulation of global gas cycles and energy conversion processes.

iridium catalysts.11 As such, CODH/ACS has potential applications for converting a potent greenhouse gas into a synthetic precursor for major industrial processes.12 Like many biological catalysts, this enzyme operates at optimal thermodynamic efficiency to be completely reversible, allowing organisms to interconvert CO2, CO, and acetyl-CoA through the Wood− Ljungdahl pathway to satisfy metabolic needs.12 Two competing mechanisms for ACS activity have emerged from prior studies. The first invokes a NiPI species that reversibly binds CO and is directly involved in acetyl-CoA formation, cycling between the NiPI, NiPII, and NiPIII states while the redox-inactive distal NiD remains in the divalent oxidation state.13,14 This proposed “paramagnetic” mechanism relies on rapid reduction of the transient NiPIII state, generated upon methyl transfer from the cobalt corrinoid protein, by a yet-unidentified redox partner. However, recent studies show that the rate of electron transfer between the coupled [Fe4S4] cluster and NiP is 200-fold slower than the rate of observed methyl transfer to NiP, casting doubt on its role in catalysis.15 Alternatively, it has been proposed that CO instead binds to a doubly reduced Ni center that cycles between the NiPII and NiP0 oxidation states.16 This “diamagnetic” mechanism is in line with that expected from synthetic organometallic nickel catalysis, though water-soluble, zerovalent nickel compounds remain a rare occurrence.17,18 In either mechanism, both CO and methyl substrates must interact with the active site of the enzyme in order for chemistry to occur, though the order of substrate binding remains unclear. The chemistry performed thus necessitates open coordination sites on the nickel center and capacity for two-electron redox processes. Controversy over the catalytic oxidation state remains in part due to the difficulty of studying the native CODH/ACS enzyme, a 300 kDa complex that is highly air-sensitive and suffers from low yields upon metal reconstitution.19,20 Studies on recombinant ACS, which lacks the CODH subunit, have also been plagued by incomplete nickel incorporation and low activities,21 though recent work has shown significant improvement on that front.22 Moreover, congestion from the [4Fe−4S] cluster and the NiD site obscures spectroscopic and computational efforts to resolve subtle changes at the NiP center.14,23 To

better understand the structure−function relationship of the ACS enzyme and elucidate the reaction mechanism, structural7,11,24−26 and functional27 mimics of the active site have been constructed using phosphine, carbene, pincer, βdiketiminate, and amide ligands.25,28−30 Intricate ligand scaffolds are required to stabilize a low-coordinate NiI state and retain the potential for substrate binding.27,31,32 However, even with the rich diversity of compounds that have been synthesized, functional mimics that reversibly generate acyl groups from gaseous CO under biologically relevant conditions have not been generated. Thus, the questions pertaining to oxidation state and catalytic mechanism remain. Previous work by our group has indicated that nickelsubstituted azurin (NiAz) may act as a simple model system for the NiP active site of acetyl-CoA synthase. Reduction of wildtype (WT) NiIIAz generates a trigonal-planar NiI active site with a similar electronic structure to a key intermediate in ACS, Ared*,33 suggesting potential for similar reactivity. However, wild-type (WT) azurin lacks a well-defined substrate binding pocket.34,35 For this reason, we sought to construct a proteinbased model that could accommodate exogenous ligands in order to develop ACS-like reactivity. The robustness of azurin toward mutations in both the primary and secondary coordination spheres renders it a favorable candidate for protein engineering.36−39 Herein, we demonstrate that the reduced, nickel-substituted M121A variant of azurin (M121A NiIAz) still closely resembles the Ared* state in ACS, supporting a trigonal planar geometry at the metal center. This construct binds carbon monoxide with physiologically relevant affinities to give a NiI−CO species that has been characterized using spectroscopic and theoretical methods. Distinct parallels are drawn between structural and spectroscopic markers of the model NiI−CO species and the CO-bound NiFeC state in ACS, and a high degree of C−O bond activation is observed in this system, a necessary criterion for acyl formation. Moreover, we show that one-electron oxidation of the protein active site gives an isolable Ni III center, an observation that is complemented by evidence for methyl binding. The ability to access three distinct oxidation states within a single metalloprotein scaffold establishes the potential for multielectron 10329


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small molecules can bind to the copper center of this variant (M121A CuIIAz).34,35,41 This reactivity was also explored within our lab, showing results similar to those previously reported. Molecules such as cyanide and thiocyanate, which are approximately isostructural to carbon monoxide, were found to bind M121A CuIIAz with dissociation constants (KD) of 12 and 4 mM, respectively (Figure S3), suggesting a reduction in the steric restrictions imposed by the protein scaffold. We note that molecules as large as acetate and phosphate are also able to bind within this cavity; as such, one must consider these effects when selecting an appropriate buffer (Figure S4). On this basis, our initial efforts focused on development of reactivity within nickel-substituted M121A azurin (M121A NiAz). Protein expression, purification, and metal substitution were carried out as previously described to yield samples containing 50− 70% nickel incorporation. As noted by others,42 we were unable to achieve quantitative metalation with nickel, perhaps due to oxidative damage of the Cys112 residue during aerobic sample handling; similar oxidative damage has previously been observed in M121G Az.43 The ligand-to-metal charge-transfer (LMCT) bands of M121A NiIIAz exhibit a hypsochromic shift relative to WT NiIIAz, with maxima at 550, 416, and 350 nm, and slightly higher extinction coefficients of 4330 and 1080 M−1 cm−1 at 416 and 350 nm, respectively (Figures S5 and S6). Addition of the one-electron reducing agent, EuIIDTPA, to M121A NiIIAz results in the appearance of a new absorption feature at higher energies (364 nm; ε = 7250 M−1 cm−1), with spectral properties that are indistinguishable from those seen for WT NiIAz (Figure S6). Considering significant rearrangement of the active site has been proposed upon reduction of WT NiIIAz, including cleavage of the methionine−nickel bond, removing the M121 residue through mutation is not expected to significantly impact the absorption spectrum of the reduced nickel center. As with WT NiIAz, aerobic reoxidation of the reduced M121A NiIAz results in near-quantitative recovery of M121A NiIIAz (Figure S7), indicating that this reduction is reversible. To further characterize the electronic structure of M121A NiIAz, CW X-band EPR spectroscopy was performed. The resultant spectrum shows a highly anisotropic, axial signal with gz = 2.56 and gx,y = 2.10 (Figure 2, Table 1), g-values that are identical to those observed for WT NiIAz and similar to those reported for the reduced Ared* state in ACS.44 The axial EPR spectrum provides further support for the proposed structure of NiIAz, a trigonal planar species in which the WT Ni−methionine bond is broken upon reduction.33 Optical and EPR potentiometric titrations were used in parallel to obtain the reported extinction coefficients for M121A NiIAz. While most properties of the M121A NiAz variant are comparable to those of WT NiAz, one primary distinction is the difference in reduction potential. It is well-known that the axial ligand in cupredoxins modulates the thermodynamics of electron transfer, resulting in potentials spanning ∼400 mV depending on the nature of that ligand.38,39,45−48 This trend is also observed in M121A CuAz, in which the CuII/I couple shifts +85−100 mV relative to WT CuAz (Figure S8).49−51 However, the NiII/I couple of the M121A NiAz variant is shifted by −39 mV relative to WT NiAz, a surprising result given prior observations that the potentials of Cu- and Ni-substituted azurin variants tend to vary in the same direction (Figure 3).38 To explain this, we consider two dominant contributing factors. Given that significant geometric reorganization is seen in the NiAz active site upon reduction for both WT and

organometallic chemistry within this simple framework. Ultimately, this research will provide fundamental insight into C1 activation processes at biological nickel centers, with implications for understanding possible reaction mechanisms of native ACS along with catalyst development and optimization.

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RESULTS AND DISCUSSION NiIAz Binds Carbon Monoxide with Low Affinity. The reduction of WT NiIIAz results in a low-coordinate nickel(I) center, mimicking the NiI-Ared* state in ACS. Following from this observation, reactivity studies were pursued with ACS substrates. Addition of carbon monoxide to WT NiIAz results in a dramatic perturbation of the EPR spectrum, with the appearance of features at gx = 2.37, gy = 2.17, and gz = 2.00 (Figure 2) that are consistent with generation of a new, nickel-

Figure 2. CW X-band EPR spectra of WT NiIAz and M121A NiIAz in the presence and absence of CO (50 mM HEPES, pH 8.0; T = 100 K; Pμw = 20 mW). Simulations are overlaid on the experimental data as dotted lines.

based species, which has been assigned to a WT NiI−CO Az. Density functional theory (DFT) calculations support the potential for CO binding to the WT NiIAz center, giving a geometry-optimized structure with lengthened metal−cysteine and metal−histidine bonds; a near-linear Ni−C−O angle of 176°; calculated electron paramagnetic resonance (EPR) gtensor values of 2.19, 2.12, and 2.03 (Table 1); and a dxz-like singly occupied molecular orbital (SOMO). However, a residual WT NiIAz signal is observed even under saturating CO concentrations, indicating weak binding affinity (Figure S1). Protein film electrochemistry (PFE) experiments performed in the presence of CO show a +15 mV shift in the WT NiII/I Az reduction potential (Figure S2), which can be converted through the Nernst equation into a dissociation constant (KD) of ∼1 mM.40 Owing to this low affinity and rapid exchange, CO vibrational signatures were not observed. These results suggest that, while methionine-121 no longer directly coordinates to the reduced nickel center, the presence of the bulky residue interferes with substrate access. Further modification of the protein will be necessary to achieve ACSlike chemistry. M121A NiIAz Closely Resembles WT NiIAz. To install a substrate-binding pocket within azurin, the aforementioned methionine-121 residue must be replaced by a smaller side chain, such as alanine (M121A Az). Prior work has suggested 10330


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Journal of the American Chemical Society Table 1. Select Spectroscopic and Calculated Geometric Properties of NiAz Modelsa WT NiII bond distance (Å)

bond angles (deg)

calc. g-values

expt. g-values

νCO(cm−1) Aiso(13C) (MHz) reduction potential (V vs NHE)

Ni−SCys Ni−CCO CCO−O Ni−C−O Ni−SCys− NHis46−NHis117 g1 g2 g3 giso gx gy gz giso expt. calc. expt. calc. expt. calc.

NiI

NiI−CO

M121A NiAz NiIII

NiII

NiI

ACS

NiI−CO

NiIII

Ared*

NiFeCb

2.169 − − − 31.70

2.165 − − − 12.85

2.322 1.792 1.151 176.00 28.91

2.295 − − − 23.16

2.175 − − − −34.23

2.177 − − − 11.01

2.316 1.796 1.145 175.17 30.37

2.262 − − − −32.97

− − − − −

2.37c 1.76 1.182d 179.10d 33e

− − − − − − − − − − − − − −

2.085 2.116 2.265 2.155 2.10 2.10 2.56 2.253 − − − − −0.593h −0.510

2.027 2.122 2.188 2.112 2.00 2.19 2.37 2.187 − 2079 − − −0.579i −

2.207 2.271 2.297 2.258 2.17 2.22 2.27 2.220 − − − − +0.877j +1.612

− − − − − − − − − − − − − −

2.106 2.123 2.289 2.173 2.10 2.10 2.56 2.253 − − − − −0.632h −0.678

2.021 2.160 2.213 2.131 2.00 2.20 2.30 2.167 1976 2090 47.6 49.2 − −

2.243 2.290 2.318 2.284 2.16 2.25 2.29 2.233 − − − − +0.826j +1.585

− − − − 2.01 2.10 2.56 2.223 − − − − − −

− − − − 2.074 2.074 2.028 2.059 1996f − 27.5g 36.1g −0.5 − −0.54 V −

a Cells with “−” reflect values that are not applicable for that species or for which information is not available. bValues from ref 22 unless otherwise stated. cAverage Ni−S bond distance. dC−O metrics from published coordinates in ref 23. eCalculated dihedral angle for M−S−S−S from PDB entry 1OAO. fFrom ref 44. gValues taken from ref 23. hReduction potentials at pH 6.0. iReduction potential at pH 5.0. jReduction potential determined from potentiometric EPR titrations at pH 5.0.

includes the active site metal, all coordinating residues, and select secondary sphere residues, including N-terminal acetylation and C-terminal amidation to give a moderately sized system for computation (Figure S9). The primary distinction between the DFT-optimized structures of WT and M121A NiIIAz lies in the fourth coordinating ligand; the M121A variant has a well-defined bond between the backbone carbonyl oxygen of G45 and nickel, similar to what is seen for M121A CuIIAz, while the WT instead shows coordination to the M121 residue (Table S1). This contributes to the changes seen in the optical spectra; time-dependent DFT (TD-DFT) calculations reproduce the hypsochromic shift in the dominant visible charge-transfer bands for the M121A variant (Figures 4 and S6). Upon reduction, the DFT-optimized structure of M121A NiIAz closely resembles WT Ni IAz, with loss of the coordinating glycine residue resulting in a trigonal planar active site (Figure S9 and Table S1). The calculated EPR gtensor values of M121A NiIAz are 2.289, 2.123, and 2.106, with 89% of the spin density localized on the Ni center in the dx2‑y2based SOMO. While, as seen with WT NiIAz, the calculated gvalue shifts are underestimated relative to the experimental values, these systematic errors are known to plague DFT calculations on d9 systems. The deviations seen here are consistent with those reported previously; however, considering relative changes in g-tensor values upon variation in redox or ligation state can still provide useful comparative information.54−57 Using a thermodynamic cycle and considering the statistical mechanical free energy of each state, reduction potential calculations were also performed (Figure S10).58,59 The NiII/I couples of WT Az and M121A are predicted to occur at −0.509 and −0.678 V vs NHE (at pH 0), respectively, which reproduces the experimental observation that the potential of the M121A NiII/IAz couple is lower than that of WT NiAz. The

Figure 3. Protein film electrochemistry of M121A NiAz. (A) Cyclic and (B) square wave voltammetry of WT NiAz (green) and M121A NiAz (black) applied onto a PGE electrode (50 mM phosphate buffer, pH 6.0, 100 mM perchlorate; ν = 200 mV/s). (Inset) Baselinecorrected voltammetric signals.

M121A NiAz (Figure S9), loss of the methionine coordination in WT would be entropically favored; an analogous favorable driving force is absent in the M121A mutant. The stronger binding of the glycine carbonyl ligand to nickel may also differentially impact the reduction potentials of the two metals. Moreover, the solvent exposure of the metal center in M121A NiAz is expected to be greater than that of WT,35 which should stabilize higher oxidation states and, correspondingly, decrease the reduction potential.51−53 In fact, the M121A CuAz potential has previously been noted to be anomalously shifted, as bulky, hydrophobic amino acid substitutions at the M121 position are typically required to shift the reduction potential in a positive direction;51 the nickel-substituted protein, by contrast, appears to follow general expectations. DFT calculations on M121A NiAz were employed to complement the experimental measurements and provide theoretical structural information on the M121A NiAz variant. A cluster model of the protein active site was constructed that 10331


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DFT calculations on a geometry-optimized structure of M121A NiI−CO Az support this assignment, with predicted gvalues and 13CO hyperfine coupling constants of 2.213, 2.160, and 2.021, and 41.2, 47.0, and 59.5 MHz, respectively. As with WT NiI−CO Az, rearrangement of the active site is seen, and a dxz-like SOMO is predicted with 89% of the spin density on the nickel center and 1.3% spin density on carbon (Figure S11). The 13CO hyperfine coupling in M121A NiI−CO Az is approximately 2-fold greater than the 13CO coupling of ∼27 MHz seen for the NiFeC state of ACS.60 However, the latter system is composed of an exchange-coupled Ni−S-[4Fe−4S] cluster, with only ∼50% of the spin density localized on the nickel center.22 This differential spin population likely accounts for the larger 13CO hyperfine coupling values observed in the model system.60 The M121A NiI−CO Az species can also be characterized using optical and vibrational spectroscopy. Addition of aliquots of CO-saturated buffer into M121A NiIAz results in a decrease of the M121A NiIAz band at 364 nm and the appearance of new features at 324 and 415 nm (Figure 6). Decomposition of this spectrum reveals that there is an additional contributing feature at 370 nm (Figure S11). Parallel EPR and optical titrations confirm that these features can be attributed to the M121A NiI−CO Az species, with extinction coefficients of ∼9800 and ∼4000 M−1cm−1 at 324 nm (31 000 cm−1) and 415 nm (24 000 cm−1), respectively. TD-DFT calculations on geometry-optimized M121A NiI−CO Az are in good agreement with these observations, predicting LMCT bands from SCys to Ni at 30 300 and 24 956 cm−1 and an MLCT band from Ni to the carbon of CO at an intermediate energy of 28 540 cm−1 (Figure S5). The calculated transition energies of M121A NiI−CO Az are within ∼1000 cm−1 of the experimental values, falling within a reasonable error for TD-DFT calculations on open-shell transition metal systems.61−67 Given the size of the model, which contains ∼70 heavy atoms and approaches the limit of what, in our hands, is computationally accessible, this deviation between experiment and theory is considered acceptable. A larger computational model that includes additional secondary sphere interactions or calculations performed at a higher level of theory would be anticipated to result in greater agreement between the computational and experimental spectra. Through EPR and optical titration experiments, a CO dissociation constant (KD) of ∼25 μM is obtained (Figure 6). This modest binding affinity suggests potential for reversible CO binding, an important feature for reactivity. For comparison, the KD values for CO binding to generate the NiFeC state of ACS range from 30−200 μM, consistent with reports of only mild CO inhibition,7,68−70 while the KD for CO binding to nickel(I) cyclam, a CO2 reduction catalyst that is known to suffer from severe product inhibition,71 is much stronger at ∼1 μM. Because of weak electrochemical signals for M121A NiAz under a CO atmosphere, protein film electrochemistry was not able to provide a complementary estimate of the CO dissociation constant.40 The nature of the Ni-CO bond was further investigated using vibrational spectroscopy and photodissociation studies. Like ACS, the M121A NiI−CO Az species is somewhat photolabile under cryogenic conditions, as visible excitation at 5 K regenerates the M121A NiIAz signal in 20% yield (Figure S13). Complete reversibility of this transition is achieved upon warming of the sample to 77 K. The yields seen here for photodissociation are directly comparable to those of ACS in

Figure 4. (A) Experimental and (B) calculated optical spectra of M121A NiIIAz (green), M121A NiIAz (black), M121A NiI−CO Az (blue), and M121A NiII−CH3 Az (orange). Experimental spectra were obtained in 50 mM HEPES, pH 8.0; calculated spectra were convolved with a Gaussian function of 2500 cm−1 line width and offset by −5000 cm−1 to facilitate direct comparison to experiment.

calculated structural, spectroscopic, and energetic properties of M121A NiIAz are consistent with the high degree of similarity to WT NiIAz and support the previously proposed geometric rearrangement of the active site upon reduction. M121A NiIAz Binds Carbon Monoxide to Model the NiFeC State of ACS. The spectroscopic and DFT results suggest M121A NiIAz features both a low-coordinate, electronrich active site and a substrate-binding pocket. To probe this hypothesis, carbon monoxide was added to M121A NiIAz. As with WT NiIAz, a dramatic change to the EPR spectrum was observed. However, in this case, complete conversion from M121A NiIAz to this new species was achieved, suggestive of quantitative binding (Figure 2). The CO-induced species has gvalues at 2.30, 2.20, and 2.00, slightly different than those observed for WT NiIAz + CO but still indicative of reorientation of the active site electronic configuration. Generation of this species using 13CO results in nearly isotropic broadening of the spectrum, with hyperfine couplings of A(13CO) = [48, 45, 50] MHz (Table S2), confirming that these features arise from a CO-bound NiI species (Figure 5A).

Figure 5. (A) CW X-band EPR spectra of M121A NiI−CO Az (blue) and M121A NiI-13CO Az (red), reduced using EuIIDTPA in 50 mM HEPES, pH 8.0 (T = 100 K; Pμw= 20 mW). (Inset) Expanded view of broadening observed at gz = 2.00. (B) Fourier transform infrared (FTIR) spectra of 500 μM M121A NiI−CO Az (blue) and M121A NiI-13CO Az (red), reduced with 15 mM EuIIDTPA in 50 mM MOPS, pH 8.0, and stirred under a CO atmosphere for 45 min. The spectrum of M121A NiIAz in the absence of CO is shown in black. 10332


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Figure 6. M121A NiAz titration with CO. (A) 15 μM M121A NiIIAz (green line) prepared in 50 mM HEPES buffer, pH 8.0. Addition of CO to M121A NiIAz (black line) shows conversion to M121A NiI−CO Az (blue). Data has been filtered to remove noise from ultraviolet−visible (UV− vis) saturation. (B) CW X-band EPR of corresponding UV−vis samples showing M121A NiIAz (black) conversion to M121A NiI−CO Az (blue). (C) Binding curve for CO binding to M121A NiIAz. The decrease of the M121A NiIAz signals (right-hand axis, black) and appearance of M121A NiI−CO Az signals (left-hand axis, blue) were monitored by UV−vis (closed circles) and EPR (open circles).

the absence of glycerol.44 FTIR spectra of M121A NiI−CO Az reveal a CO stretching frequency of 1976 cm−1 that shifts to 1932 cm−1 when 13CO is used, consistent with that expected for an isolated harmonic mode (Figure 5B). DFT vibrational frequency calculations on the optimized structure agree with the experimental stretching frequency (Figure S14) and suggest a C−O bond distance of 1.145 Å, suggestive of an activated CO moiety relative to the free gaseous molecule. The degree of C− O bond activation in M121A NiI−CO Az is comparable to those seen in the native ACS enzyme, which exhibits a νCO of 1996 cm−1,44 and even the active species in the industrial Monsanto and Cativa processes, which feature CO stretching frequencies of 1981/2065 and 1970/2048 cm−1, respectively.72,73 Coupled with the modest binding constant, these data highlight the potential for downstream chemistry to act upon the bound carbonyl species. The comprehensive spectroscopic characterization of M121A NiI−CO Az highlights the similarities between the model protein system and the NiFeC state of ACS (Table 1), underscoring potential for achieving related reactivity. NiAz is Capable of Accessing Three Distinct Oxidation States. ACS formally participates in organometallic chemistry, undergoing multicomponent, two-electron oxidative addition and reductive elimination reactions. This is distinct from the typical reactivity seen in hydrogenase, superoxide dismutase, and CODH, in which nickel undergoes proton-coupled, oneelectron transfer processes. In typical model compounds, accessing both the II/I and III/II couples is physiologically unattainable.24 However, the nickel site in ACS must pass through three oxidation states, even if some are only transiently formed. In the paramagnetic mechanism, for example, a NiIII− CH3 species is implicated upon methyl transfer from the corrinoid protein. However, under in vitro assay conditions, this high-potential state seems to be rapidly reduced to a NiII−CH3 species. Thus, only indirect evidence exists for the multielectron capacity of the ACS active site, a point that underlies the ongoing controversy about the ACS mechanism. To directly probe the potential for this chemical reactivity, we sought to investigate whether the NiAz systems could access a NiIII oxidation state using the one-electron oxidant Na2IrCl6. The addition of Na2IrCl6 to WT NiAz followed by rapid freezing results in an almost isotropic EPR signal, with experimental gvalues of 2.27, 2.22, and 2.17 and a substantial amount of anisotropic broadening (Figure 7). The significant deviation in energy from the free-electron g-value of 2.0023 indicates a large

Figure 7. CW X-band EPR spectra at 100 K of WT (gray) and M121A (purple) NiIIIAz, generated using 2 mM Na2IrCl6 in 50 mM acetate, pH 4.5 (WT) or 50 mM Na2IrCl6 in 50 mM MES, pH 5.0 (M121A). Spectral simulations (dotted lines) are overlaid on experimental data. (Inset) Power saturation curves of (left) WT and (right) M121A NiIIIAz signal (T = 5 K).

degree of spin−orbit coupling, suggesting the signal arises from a nickel-centered species, and control experiments confirm that this signal is protein-derived (Figure S15). No additional features are seen at other fields or low temperatures that would suggest the presence of a high-spin species (Figure S16). Power dependence experiments at low temperatures indicate that the observed signal saturates using fairly low powers (Figure 7, inset, P1/2 = 0.127 mW at 5 K), as would be expected for an isolated, low-spin d7 system; high-spin, S = 3/2 systems typically demonstrate rapid relaxation and thus require high powers for signal saturation.74 An analogous experiment was performed with M121A NiIIAz, resulting in an approximately isotropic but slightly different EPR signal with g-values of 2.29, 2.25, and 2.16 and unresolved broadenings due to strain (Figure 7). The observed M121A NiIIIAz saturates with a P1/2 = 0.175 mW at 5 K. With both systems, a large degree of spin−orbit coupling is observed, suggestive of a nickel-centered species with the unpaired electron in the dx2‑y2 or dxy orbital. The observed relaxation 10333


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Journal of the American Chemical Society properties of both signals are consistent with those of S = 1/2 systems in distorted tetrahedral geometries. Potentiometric EPR titrations indicate low yields and high equilibrium potentials of +0.877 and +0.826 V vs NHE for WT and M121A NiIIIAz, respectively (Figure S15). DFT calculations support this analysis, giving predicted g-tensors in good agreement with experiment and calculated reduction potentials consistent with this trend (Table 1). In the case of M121A NiIIIAz, the empty cavity may support binding of a water molecule, though deprotonation to hydroxide appears unlikely on the basis of the calculated g-tensors (Table S3). Advanced EPR experiments and computational analyses are underway to further characterize these high-valent species. However, the ability of NiAz to access both the II/I and III/II couples under physiological conditions highlights its potential use for bioorganometallic chemistry. M121A NiIAz Shows a Weak Interaction with a Methyl Donor. Given the demonstrated multielectron capacity of M121A NiAz, we were interested in exploring reactivity with organic species. The second ACS substrate is a methyl group, which, in the native system, is donated by the CoIII−CH3 corrinoid cofactor of an associated subunit. While no changes were observed upon addition of methyl-cobinamide or methylcobalamin to M121A NiIAz, likely owing to steric constraints, the addition of methyl iodide (CH3I) as a methyl donor perturbed both the optical and EPR spectra. As with the addition of CO, the NiIAz optical band at 364 nm decreased, with concomitant growth of bands at 416 and 324 nm (Figure 8A) that are suggested to arise from the interaction with −CH3 (Figure S17) rather than iodide or other solution components. Titration experiments reveal a dissociation constant (KD) for CH3I binding to M121A NiIAz of ∼100 μM (Figure 8A, inset), and recovery of the M121A NiIIAz spectrum was seen upon reoxidation, again suggesting a reversible binding process. The NiIAz EPR signal also decreased upon addition of CH3I, though, surprisingly, no new features were observed that could be attributed to a NiIII−CH3 species (Figure S18). To explain this absence, we note that, like experiments performed on ACS itself, the reaction conditions require the presence of excess reducing agent in solution to generate and maintain the NiIAz state. Thus, upon binding of a methyl group, the high-potential NiIII−CH3 Az active site is likely rapidly reduced to a NiII−CH3 state. This process is analogous to that proposed for the native ACS system, in which a NiIII−CH3 state has not yet been isolated. DFT investigations on a geometry-optimized NiII− CH3 model show good agreement between the calculated and experimental optical spectra (Figures 4 and S19), and further characterization studies of this putative organometallic species are underway in our laboratory. To investigate the differential binding affinities of CO and −CH3 groups to M121A NiIAz, competition experiments were performed with both substrates (Figure 8B). In one experiment, CH3I was added first to a solution of M121A NiIAz. An aliquot removed for EPR measurement showed complete quenching of the EPR signal. The remaining sample was subsequently exposed to a CO atmosphere, and the characteristic M121A NiI−CO Az signal was recovered, albeit with lower intensity due to slow metal loss from the active site. When substrates were added to M121A NiIAz in the reverse order, the M121A NiI−CO Az signal persisted, even in the presence of excess CH3I (Figure S20). These observations indicate that M121A NiIAz exhibits a higher binding affinity for CO than −CH3 groups. Interestingly, −CH3 was unable to bind to WT

Figure 8. M121A NiAz titration with CH3I. (A) 15 μM M121A NiIIAz (green line) prepared in 50 mM HEPES buffer, pH 8.0. Addition of CH3I to M121A NiIAz (black line) shows conversion to a new, EPRsilent species (orange). Data has been filtered to remove UV−vis noise at higher energies. (Inset) Binding curve for −CH3 binding to M121A NiIAz. (B) CW X-band EPR of reduced M121A NiIAz (black trace) to which CH3I was first added (orange trace), followed by addition of CO (blue trace).

NiIAz, perhaps due to the steric restrictions of the methionine residue (Figure S21). Implications for the Mechanism of ACS. To the best of our knowledge, NiAz is the first model nickel protein capable of accessing three oxidation states within a physiological range. Even synthetic nickel compounds that can exist in three isolable oxidation states in organic solvents are rare, as the NiII/I and NiI/0 reduction potentials are typically inverted, resulting in thermodynamically favorable two-electron reduction processes.75 On the other end, the highly oxidizing NiIII system is generally stabilized by employing either redox noninnocent ligand frameworks that move the hole onto the ligand center or highly fluorinated ligands to withdraw electron density. However, the combined thiolato-, bis-imidazole coordination of the Az active site appears to provide the electronic structure necessary to support both the reduced and oxidized species. The NiIAz state binds CO, an ACS substrate, with physiologically relevant binding affinity. This NiI−CO Az state closely resembles the NiFeC state, which recently has been shown to be an intermediate for ACS catalysis.22 Moreover, M121A NiIAz shows weaker binding affinity for −CH3 groups than for CO, which has implications for addressing the order of substrate binding in the native ACS system. While carbon−carbon bond formation and acyl transfer remain to be demonstrated in this engineered protein and represent ongoing efforts in our laboratory, the work presented here establishes a foundation for developing enzymatic ACSlike chemistry using the NiIII, NiII, and NiI oxidation states, as a 10334


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Figure 9. Comparison of paramagnetic ACS mechanism and putative M121A NiAz mechanism. Intermediates for which a NiAz-based model has been made are highlighted in colored circles. Steps of the putative catalytic cycle for NiAz that have not yet been observed are shown as dotted lines. NHE). Fresh Na2IrCl6 solutions were prepared immediately prior to use. Optical Spectroscopy. Absorption spectra were measured on a Shimadzu UV-2600 spectrophotometer. WT NiIIAz and M121A NiIIAz samples were diluted into 50 mM HEPES buffer (GoldBio), pH 8.0. WT NiIAz and M121A NiIAz samples were prepared under a nitrogen atmosphere in an anaerobic glovebox (Vigor Technologies) with gastight cuvettes. For the titration of M121A NiIAz with CO, an aliquot of 50 mM HEPES buffer, pH 8.0 was first sparged with CO until saturated. The concentration of a saturated CO solution was determined to be ∼1 mM CO. During experiments with carbon monoxide, CO gas was kept continuously flowing through a septum-capped vial. Aliquots of sample were removed using a gastight syringe and transferred to a septumcapped cuvette inside an anaerobic glovebox. Determination of the extinction coefficients for M121A NiIAz and M121A NiI−CO Az was accomplished using both UV−vis and EPR spectroscopy in tandem. Samples that showed complete optical conversion of M121A NiIIAz to M121A NiIAz or M121A NiI−CO were quantified in EPR against a known CuAz standard measured under nonsaturating conditions (T = 100 K; Pμw = 20 mW). This concentration was then used along with the absorbance to determine the extinction coefficient. These values are subject to ∼10% error due to the uncertainty in spin-quantitation of samples.51 Optical titrations with CH3I were accomplished by anaerobically preparing a 10 mM solution of CH3I in ethanol. After reduction with EuIIDTPA, aliquots of this stock solution of CH3I were added to a stock solution of ∼15 μM protein. At each titrant addition step, optical spectra were recorded in a septum-capped cuvette, and a 150 μL sample was removed for EPR measurement. The KD of −CH3 binding was determined by monitoring the decrease of M121A NiIAz in both UV−vis and EPR until no further decrease was observed and assuming 100% conversion to a −CH3 bound species at that point. Electrochemistry. All electrochemical experiments were carried out as previously described within a nitrogen glovebox.33 Specific electrochemical parameters are included in the Supporting Information. All reported potentials were converted from the Ag/AgCl reference electrode used for measurement to the normal hydrogen electrode (NHE) by the addition of +0.198 V. Analysis of resulting voltammograms was performed using the SOAS program.80 Electron Paramagnetic Resonance Spectroscopy. Continuous-wave (CW) X-band EPR spectra at 100 K were collected using a Bruker EMXPlus equipped with a Bruker variable temperature unit. CW X-band EPR spectra measured at 5 K were collected at the Ohio Advanced EPR Facility at Miami University using a Bruker EMX instrument equipped with an Oxford flow cryostat (ITC-500). WT NiIAz samples were prepared as previously described;33 M121A I Ni Az samples were prepared by the addition of EuIIDTPA to a 500 μM protein solution prepared in 50 mM HEPES, pH 8.0. For EPR samples containing CO, the reduced protein was left to stir in a 20 mL septum-capped vial under a continuously flowing CO atmosphere for 45 min. Samples containing 13CO were prepared in a similar way; however, the vial was backfilled with 13CO to retain positive pressure instead of incubating under continuously flowing 13CO. After being

significant fraction of critical ACS intermediates have now been modeled in the simple NiAz protein system (Figure 9).

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CONCLUSIONS We have shown that NiAz can access multiple oxidation states within a single protein scaffold without the aid of noncanonical amino acids or post-translational modifications, rendering this model system a promising candidate for performing facile, twoelectron processes. Additionally, the WT NiAz scaffold can be modified to install an active site capable of binding the ACS substrates CO and −CH3. Using diverse types of spectroscopy coupled with DFT calculations, the NiI−CO species has been characterized. This species exhibits strong similarity to the NiFeC state in the native A-cluster, serving as an effective model of this key intermediate of ACS. Evidence is also presented demonstrating an interaction between CH3I and M121A NiIAz, with competition experiments indicating weaker binding affinity for the −CH3 group than CO. Taken together, this work highlights the utility of the NiAz system as a model for the NiP site in acetyl-CoA synthase, with implications for better understanding the native enzyme system as well as potential for guiding design of inexpensive and robust nickelbased catalysts for efficient carbon−carbon bond-coupling reactions.

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EXPERIMENTAL SECTION

All materials were used as received unless otherwise noted. Solutions were prepared using deionized water (18.2 MOhm, Elga Technologies) unless otherwise noted. Protein Expression, Purification, and Metal Substitution. Pseudomonas aeruginosa wild-type and M121A azurins were heterologously expressed, purified, and metalated as described previously.33,37,52,76−78 Nickel incorporation was achieved near-quantitatively into WT Az and to a level of ∼60−70% in the M121A mutant (Figure S7). Preparation of EuIIDTPA. Preparation of europium(II) diethylene triamine pentaacetic acid (EuIIDTPA) stock solution was carried out in two ways. The first method (method one) followed a previously published protocol.79 Alternatively (method two), to exclude excess Cl− ions, EuIIDTPA was prepared by direct addition of EuIICl2 to a 100 mM DTPA solution at pH 7.0, made in a 1:1 1 M NaOH:H2O solution, resulting in a 100 mM stock solution of EuIIDTPA. The potential of the EuIIDTPA stock solution was measured using a twoelectrode system with a graphite working electrode and Ag/AgCl reference and found to be −1.10 V vs Ag/AgCl (−902 mV vs NHE). Fresh EuIIDTPA stock solutions were always made immediately prior to sample preparation. Preparation of Na2IrCl6. Sodium hexachloroiridate(IV) (Alfa Aesar) was prepared as a 50 mM solution in either 50 mM MES (GoldBio), pH 5.0, or 50 mM sodium acetate (Amresco), pH 4.5. Resulting reduction potentials were 0.780 V vs Ag/AgCl (0.978 V vs 10335


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Journal of the American Chemical Society stirred, the samples were transferred into EPR tubes (Wilmad LabGlass 727-SQ-250MM) and frozen in liquid nitrogen. For preparation of NiIII EPR samples, an aliquot of 50 mM Na2IrCl6 was added to the protein sample, and the potential was measured using a two-electrode system and a voltmeter with a Pt wire working electrode and a Ag/AgCl reference electrode. Because of decreased stability of NiAz and M121A NiAz at low pH, samples were prepared, potentials were measured, and samples were frozen within ∼2 min. EPR spectra were acquired for approximately 45 min using a microwave power of 20 mW at 100 K. A modulation frequency and amplitude of 100 kHz and 10 G, respectively, were used for all spectra. Power saturation studies were performed and analyzed as described previously.33,81 The spectra were corrected for residual reducing agent or oxidizing agent baseline signals by subtraction of a spline using Igor Pro (Wavemetrics, Lake Oswego, OR) data analysis software. EPR spectral simulations were carried out using the EasySpin toolbox within MATLAB.82 Fourier-Transform Infrared Spectroscopy. All FTIR samples were prepared anaerobically inside a nitrogen glovebox atmosphere. A 500 μM M121A NiIIAz protein sample was prepared in 50 mM MOPs, pH 8.0, reduced with 15 mM EuIIDTPA, and left under a CO atmosphere, stirring, for 45 min. A 12CO atmosphere was achieved by continuously flowing natural-abundance CO through a septum-capped GC vial. Samples containing 13CO were prepared in a similar fashion, with the vial under positive 13CO pressure rather than a continuously flowing atmosphere. After 45 min, 40 μL of sample were transferred to an anaerobic FTIR cell with CaF2 windows (Thermo Fisher Scientific) and a 0.05 mm Teflon spacer. The sample spectrum was averaged over 500 scans using a Bruker Tensor 27 FTIR instrument with 2 cm−1 resolution. Spectra were corrected for broad background signals by subtraction of the spectrum of EuIIDTPA in 50 mM MOPS, pH 8.0. Density Functional Theory Calculations. All calculations were carried out using the computational chemistry software package ORCA83 and analyzed using the ChemCraft program (www. chemcraftprog.com). A truncated model of the M121A NiAz active site was constructed using the published crystal structure for nickelsubstituted Pseudomonas aeruginosa azurin (PDB entry 1NZR) and replacing the methionine-121 residue with alanine using the mutagenesis wizard in PyMol.84 The residues coordinated to the metal center (G45, H46, C112, H117, M121A) were taken in their entirety along with N47. Each noncontinuous side chain was terminated with N-acetylation and C-amidation to retain charge neutrality. This model was used in all calculations. The Cartesian coordinates of all backbone carbonyl carbon atoms were constrained to retain the fold of the protein, and only the S = 1 spin state was considered for the M121A NiIIAz species. Geometry optimizations were first carried out using the BP86 functional with the RI approximation applied followed by use of the B3LYP functional using the RIJCOSX approximation.85 All calculations used the def2TZVP basis set for nickel and all directly coordinated atoms and a def2-SV(P) basis set for all other atoms.86 To model the dielectric effects of an enzyme environment, COSMO was invoked during geometry optimizations87 until an energy minimized geometry was obtained. At this point the geometry was then further optimized in the gas phase. The gas-phase optimized geometry was used for all further analysis. The geometry-optimized M121A NiIAz or M121A NiIIIAz models were generated using the methods above, modifying the charge and multiplicity. For the M121A NiI−CO Az, M121A NiII−CH3 Az, M121A NiIII−OH Az, and M121A NiIII−H2O Az models, addition of a CO, −CH3, −OH, or H2O ligand to the metal center was achieved using the ChemCraft software. The geometry-optimized structure of WT NiI−CO Az structure was constructed by the addition of a CO ligand in ChemCraft to a previously optimized model33 followed by rounds of reoptimization. Similarly, optimization of the WT NiIIIAz model was carried out by modifying the charge and multiplicity of the system and reoptimizing the structure. Only the S = 1/2 state was considered for the NiIIIAz models owing to the experimental g-tensor and slow relaxation properties, which are consistent with a low-spin Ni-centered species.88

The geometry-optimized structures and the B3LYP functional were used to calculate EPR g-tensors, hyperfine couplings, spin densities, molecular orbitals, and vibrational frequencies.89−91 Prior work has shown the NiAz EPR g-tensor calculations to be independent of functional.33 Optical absorption spectra of M121A NiIIAz, M121A NiIAz, M121A NiI−CO Az, and M121A NiII−CH3 Az were calculated via TD-DFT in the gas phase. To investigate the functional dependence on optical transition energies and intensities, the use of different DFT functionals was attempted (Figure S23).61−63,67,92 The spectra calculated with the B3LYP functional were found to be the most representative of trends seen across the entire suite of redox and ligand states for NiAz. Vibrational frequencies were calculated in the gas phase. Spin densities and molecular orbitals were generated and visualized using the orca_plot module, while TD-DFT results were visualized using the orca_mapspc module.83 A sample input file and all optimized coordinates are given in the Supporting Information. The reduction potentials of the WT NiII/IAz and M121A NiII/IAz couples were calculated using a thermodynamic cycle and considering the free energy of each state in solution and in the gas phase (Figure S9).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b03892. Detailed materials and methods, supplemental simulations and EPR spectra, optical titrations, DFT structures and coordinates, details on reduction potential calculations, and electrochemical voltammograms of M121A CuAz (PDF)

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AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Hannah S. Shafaat: 0000-0003-0793-4650 Present Address †

M.J.O.: School of Education, University of Wisconsin Madison, 225 N Mills St, Madison, WI 53706. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge Professor Yi Lu for generous donation of the pET9a M121A plasmid, Dr. Robert McCarrick at the Ohio Advanced EPR Facility at Miami University, Professor David Grahame for donation of a sample of methyl cobinamide, Dr. Michael Stevenson and Sean Marguet for helpful discussions, and Jeffrey Slater for help with data collection. This work has been supported by the OSU Department of Chemistry and Biochemistry and the ACS PRF Fund (57403-DNI6). C.R.S. acknowledges support from an NIH Chemistry-Biology Training Grant (GM-08512) and M.J.O. acknowledges the OSU College of Arts and Sciences Honors Undergraduate Research Scholarship.

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REFERENCES

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