Structural and Functional Relevance of the Conserved Residue V13 in

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Structural and Functional Relevance of the Conserved Residue V13 in the Triheme Cytochrome PpcA From Geobacter Sulfurreducens Joana M Dantas, Pilar C. Portela, Ana P. Fernandes, Yuri Y. Londer, Xiaojing Yang, Norma E.C. Duke, Marianne Schiffer, Phani Raj Pokkuluri, and Carlos A. Salgueiro J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b01214 • Publication Date (Web): 15 Mar 2019 Downloaded from http://pubs.acs.org on March 19, 2019

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The Journal of Physical Chemistry

Structural and Functional Relevance of the Conserved Residue V13 in the Triheme Cytochrome PpcA from Geobacter sulfurreducens Joana M. Dantas1, Pilar C. Portela1, Ana P. Fernandes1, Yuri Y. Londer2, Xiaojing Yang2†, Norma E. C. Duke2†, Marianne Schiffer2, P. Raj Pokkuluri 2,* Carlos A. Salgueiro1,* 1

UCIBIO-Requimte, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de

Lisboa, Campus Caparica, 2829-516 Caparica, Portugal. 2

Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, Illinois 60439,

USA.

*

Corresponding authors: [email protected] and [email protected]

†Present address: Norma Duke, SER-CAT and the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA. Xiaojing Yang, Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, USA.

The submitted manuscript has been created by UChicago Argonne, LLC, Operator of Argonne National Laboratory (“Argonne”). Argonne, a U.S. Department of Energy Office of Science laboratory, is operated under Contract No. DE‐AC02‐06CH11357. The U.S. Government retains for itself, and others acting on its behalf, a paid‐up nonexclusive, irrevocable worldwide license in said article to reproduce, prepare derivative works, distribute copies to the public, and perform publicly and display publicly, by or on behalf of the Government.

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ABSTRACT The triheme cytochrome PpcA from Geobacter sulfurreducens is highly abundant under several growth conditions and is important for extracellular electron transfer. PpcA plays a central role in transferring electrons resulting from the cytoplasmic oxidation of carbon compounds to the cell exterior. This cytochrome is designed to couple electron and proton transfer at physiological pH, a process achieved via the selection of dominant microstates during the redox cycle of the protein, which are ultimately regulated by a wellestablished order of oxidation of the heme groups. The three hemes are covered only by a polypeptide chain of 71 residues and are located in the small hydrophobic core of the protein. In the present work, we used NMR and X-ray crystallography to investigate the structural and functional role of a conserved valine residue (V13) located within van der Waals contact of hemes III and IV. The residue was replaced by alanine (V13A), isoleucine (V13I), serine (V13S) and threonine (V13T) to probe the effects of the side chain volume and polarity. All mutants have been found to be as equally thermally stable as the native protein. The V13A and V13T mutants produced crystals and their structures were determined. The side chain of threonine residue introduced in V13T showed two conformations but otherwise the two structures did not show significant changes from the native structure. Analysis of the redox behavior of the four mutants showed that for the hydrophobic replacements (V13A and V13I) the redox properties, and hence the order of oxidation of the hemes was unaffected in spite of the larger side chain, isoleucine, showing two conformations with minor changes of the protein in the heme core. On the other hand, the polar replacements (V13S and V13T) showed the presence of two conformations more distinctly and the oxidation order of the hemes was altered. Overall, it is striking that a single residue with proper size and polarity, V13, was naturally selected to assure a unique conformation of the protein and the order of oxidation of the hemes, endowing the cytochrome, PpcA, the optimal functional properties necessary to assure effectiveness in the extracellular electron transfer respiratory pathways of G. sulfurreducens.

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INTRODUCTION Membrane electron transfer chains have a central role in cellular life. With the exception of photosynthesis in which light-driven charge separation is necessary to extract electron from water molecules, the electron transfer in the respiratory chains typically occurs as sequential electronic reactions from low-redox potential donors to high-redox-potential electron acceptors. In eukaryotic cells, the electrons resulting from oxidation of carbon sources are injected into the respiratory chain by the low-redox potential donor nicotinamide adenine dinucleotide hydride (NADH) and/or flavin adenine dinucleotide (FADH2). These electrons are then transferred to a series of components that are either intimately associated with the mitochondrial innermembrane (eg. NADH dehydrogenase or cytochrome c oxidase), soluble in the mitochondrial intermembrane space (eg. cytochrome c) or freely-diffusing within the phospholipid layer (eg. quinones). This respiratory chain culminates with the reduction of oxygen to water and the energy released by the successive electron transfer steps contributes to the formation of a proton electrochemical gradient at the inner mitochondrial membrane and concomitant production of ATP by ATP synthases. Although using a similar strategy and molecular electron transfer components, the respiration processes in prokaryotes are much more versatile. For example, the high-redox-potential terminal electron acceptor oxygen can be replaced by a diverse range of soluble or insoluble electron acceptors, which have an impact on the cellular topology of the respiratory electron transfer components. In the case of soluble electron acceptors that can readily diffuse to periplasm, the topology resembles that of the mitochondrial respiratory chain with electron transfer components associated to the cytoplasmic membrane or soluble in the periplasm. However, when the acceptors are unable to freely diffuse into the cells, their reduction requires transfer of electrons to the cell exterior and, therefore, a different arrangement of the electron transfer proteins.1, 2 Shewanella and Geobacter bacteria are well-studied examples of microorganisms capable of reducing insoluble metal oxides and other extracellular electron acceptors, such as electrode surfaces from which electric current can be harvested.3 The reduction of external acceptors, by a mechanism known as extracellular electron transfer (EET), is of special interest for biotechnological applications, particularly in the bioremediation of toxic or radioactive metals and bioenergy production by microbial fuel cells.4-7 To assure EET, microorganisms have evolved so that the cellular organization of the electron transfer components differs from those microorganisms unable to utilize extracellular acceptors. In fact, in microorganisms capable of EET, electron transfer components are associated to the outer membrane, in addition to the common location in the inner membrane and periplasmic space.1, 8 The genetic systems developed for these two bacteria coupled to several genetic and proteomic studies have identified key c-type cytochromes for EET (for a review see9,

10

). In the particular case of G.

sulfurreducens, which is known as the most efficient exoelectrogenic bacterium with natural occurrence in several environments6, the present model for EET considers that upon reduction by NADH-dehydrogenase of freely-diffusing quinones within the phospholipid layer, electrons are transferred to periplasmic cytochromes by two different inner membrane associated cytochromes, CbcL or ImcH. The predominance of one or another electron transfer pathway was proposed to be dependent on the redox potential of the terminal 3



electron acceptors.11,

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In both cases, after reaching the periplasmic components, the electrons are then

transferred toward outer membrane cytochromes before reaching the extracellular terminal acceptors. In addition to the electron transfer components described above, G. sulfurreducens cells also developed microbial nanowires, designated by pili, which are electrically conductive filaments that facilitate long-range EET to Fe(III) oxides and high-density current production in microbial fuel cells.13 The periplasmic cytochromes, and particularly the triheme cytochrome PpcA from G. sulfurreducens, is always present to guarantee an efficient interface between the cytoplasm and outer membrane components.14, 15

Structural and functional data have been obtained for cytochrome PpcA.14, 16-19 The three heme groups are

bis-histidinyl axially coordinated and have negative but different redox potentials. The heme redox potentials are either modulated by redox and redox-Bohr interactions. The first measures the effect of the oxidation state of the heme neighbors on a particular heme redox potential whereas the redox-Bohr interactions account for the modulation of the solution pH on the heme redox potential values. The fine-tuned network of hemes’ redox interactions has revealed that the protein possesses the necessary characteristics to transfer both electron and protons and to contribute to the formation of a proton electrochemical gradient across the periplasmic membrane. The regulation of this network is highly dependent upon the microscopic properties of the heme groups and hence on their order of oxidation.20-22 Thus, to understand and further improve the EET processes, it is important to elucidate the role of key residues that endow PpcA with the necessary structural and functional properties. The side chain of residue valine 13 (V13) is placed within van der Waals distance between hemes III and IV from PpcA.16 This residue, as well as phenylalanine 15 (F15), the side chain of which is placed in between hemes I and III, are integral part of the hydrophobic heme core of the protein and are located on the second β-strand of the protein structure (Fig. 1). Whereas F15 is highly conserved in the cytochrome c7 and c3 families, V13 is observed to be replaced by isoleucine in a few cases (accession numbers for two examples: WP_004512000 and OGT99157). Previously, we reported the effects of changing the highly conserved F15 on the structural and functional properties of PpcA.23,

24

In the present work, we report our observations on the single site

substitutions of V13 by hydrophobic residues (alanine and isoleucine) and by polar (serine and threonine) residues. Based on the structure of PpcA, it was expected that isoleucine at position 13 could not be accommodated without a significant perturbation in the local structure. On the other hand, serine and threonine are expected to be accommodated within the spatial constraints but it is of interest to study the effects of introducing a polar group within the hydrophobic environment of the heme core and on the redox properties of PpcA. MATERIALS AND METHODS Production of PpcA mutants The oligonucleotides for the PpcA mutants (V13A, V13I, V13S and V13T) were designed by the QuikChange Primer Design program (Agilent Technologies) and synthesized by MWG Biotech (High Point, NC). Then the QuikChange Site-Directed Mutagenesis Kit (Stratagene) was used to produce the modified 4


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genes using as template the PpcA expression vector pCK32.25 The presence of desired mutations was confirmed by DNA sequencing in both strands by MWG Biotech. Production of PpcA mutants Escherichia coli cells [strain BL21(DE3)] harboring the plasmid pEC86, which contains the genes for the maturation of c-type cytochromes26, were transformed with either one of the expression vectors containing PpcA mutated sequences (V13A/I/S/T). The cells were then grown aerobically at 30°C in 2xYT medium, containing 34 µg/mL chloramphenicol and 100 µg/mL ampicillin, to mid-exponential phase at 200 rpm to an OD600 of approximately 1.5. The protein expression was then induced with 10 μM of isopropyl β-Dthiogalactoside and incubated overnight at 30°C and 180 rpm after which the cells were harvested. The cell pellets were gently resuspended in 60 ml of ice-cold TES buffer (100 mM Tris-HCl (pH 8.0), 0.5 mM EDTA and 20% sucrose) containing lysozyme (0.5 mg/mL per liter of inicial culture). The periplasmic fraction was recovered by centrifugation at 14700 xg, at 4 C for 20 minutes. The supernatant constituted the periplasmic fraction, which was ultracentrifuged at 225000 xg, at 4 C for 1h and then dialyzed against 4.5 L of 10 mM Tris-HCl pH 8.5. Each mutant was purified by ionic exchange and gel filtration, as described for the wild-type cytochrome.27 Briefly, each periplasmic fraction was loaded onto 2x 5 ml Econo-Pac High S cartridges (BioRad) equilibrated with 10 mM Tris HCl pH 8.5 and the protein was eluted with a 0.3-1 M NaCl gradient at a flow rate of 1 mL/min. The fractions with higher purity index (determined by the ratio of the absorbances measured at 408 nm and 210 nm) were combined, concentrated to approximately 1 ml, loaded onto a Superdex 75 molecular exclusion column (GE Healthcare), and eluted with 100 mM sodium phosphate buffer, pH 8 at a flow rate of 0.5 mL/min. The purity of the proteins was evaluated by SDS-PAGE (15%) stained with Coomassie blue. Crystallization Purified concentrated protein in 20 mM sodium phosphate buffer pH 7.8 with 100 mM NaCl was used for crystallization trials by hanging drop method at room temperature. Several conditions close to the native PpcA27 were explored to identify crystals for the V13 mutants. Two mutants, V13A and V13T, produced diffraction quality crystals but the other V13 mutants, V13S and V13I, did not produce crystals in the conditions close to the native protein. We did not pursue crystallization of V13S and V13I in a wider search because of our previous experience with PpcA in which extensive crystallization trials failed to produce crystals in conditions other than the published conditions that include the additive deoxycholate. In case of V13A the protein concentration was 13 mg/mL. The crystal used for data collection was retrieved from the following conditions. Reservoir consisted of 0.7 mL of 3.7 M ammonium sulfate pH adjusted to 6; drop is 1 µl protein and 1 µl reservoir plus 0.5 µl of 0.25% (w/v) deoxycholic acid sodium salt. Cryo protection was achieved by transferring the crystal briefly to a solution of 3.5 M ammonium sulfate containing 28% sucrose. For V13T the protein concentration was 23 mg/mL. The crystal used was obtained

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from a drop consisting of 1 µl protein and 1 µl reservoir plus 0.5 µl of 0.25% (w/v) deoxycholic acid sodium salt equilibrated over a reservoir made up of 0.7 mL of 3.5 M ammonium sulfate pH adjusted to 6. The crystal was cryoprotected as mentioned above for V13A mutant. Data collection and structure determination The X-ray diffraction data was collected at the 19BM beam line of the Structural Biology Center, Advanced Photon Source (Argonne, IL). Data reduction and scaling was achieved with the program HKL 200028. The crystals of the two mutants were isomorphous with the native PpcA crystals. Structure solution was achieved by rigid body refinement using the native coordinates (PDB code, 1OS6), after removing the side chain atoms beyond C of V13 using the program CNS29. Refinement was carried out by the program Refmac30 with intermittent examination of the electron density maps for model adjustments and adding solvent molecules as needed were done by the program Chain31. The final data collection and refinement statistics are presented in Table 1. NMR studies Preparation of NMR samples In order to match the experimental conditions previously used for the wild-type cytochrome,14 PpcA valine mutant samples were prepared with the following concentrations: 140 µM for studies carried out in the reduced form and 70 µM for NMR redox titrations. Protein samples were firstly lyophilized and then resuspended in 80 mM phosphate buffer with NaCl (250 mM final ionic strength) in 2H2O. Each NMR tube was sealed with a gas-tight serum cap and reduction of the samples was obtained by the catalytic reduction with the enzyme Fe-hydrogenase from Desulfovibrio vulgaris (Hildenborough) in the presence of gaseous hydrogen, as previously described for the wild-type protein. 14 For the NMR redox titrations, the samples were firstly reduced and then hydrogen was replaced with argon. Once excess of hydrogen was removed, the samples were slowly oxidized to the desired intermediate level of oxidation by the addition of controlled amounts of air into the NMR tube. To assure that samples are stable at the chosen intermediate oxidation level, the pH was measured inside an anaerobic glove chamber, as previously described for the wild-type cytochrome. 14

NMR experiments and assignment NMR experiments were performed on a Bruker Avance III 600 spectrometer at 288 K matching those previously obtained for PpcA cytochrome.14, 32 2D 1H-TOCSY and NOESY were acquired with 45 and 100 ms mixing-time, respectively, to assist the assignment of the heme substituents signals in the fully reduced proteins. For partially oxidized samples, 2D 1H-EXchange SpectroscopY (EXSY) spectra were acquired to monitor the stepwise oxidation of the individual hemes in the four V13 mutants.

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The NMR spectra were processed using TOPSPIN (BrukerBiospin, Karlsruhe, Germany). The signals were assigned with the program Sparky - NMR Assignment and Integration Software33 following the methodology previously described.14, 32 RESULTS AND DISCUSSION Effect of the mutations on the cytochrome’s fold One of the hallmarks of the cytochrome PpcA is the highly compact arrangement of the three heme groups within the small hydrophobic core of the protein (Fig. 1). This is a consequence of the relatively short polypeptide chain, with only 71 amino acids, to cover the three hemes. This compact arrangement provides a very particular chemical environment to the heme core so that the observed heme substituent 1H NMR signals are essentially determined by their relative orientation and neighbouring residues. In fact, because the hemes are low-spin in the reduced state, typical regions for the heme substituent signals are observed: 8 to 10 ppm for meso protons (5H, 10H, 15H and 20H, accordingly to the IUPAC nomenclature for tetrapyrroles that is illustrated in Supplementary Fig. S1); 6 to 8 ppm for thioether methines (31H and 81H); 2.5 to 5 ppm for methyl groups (21CH3, 71CH3, 121CH3 and 181CH3); and -1 to 3 ppm for thioether methyls (32CH3 and 82CH3). In the present work, 2D 1H NMR spectra (NOESY and TOCSY) were used to assign the heme substituent signals in each mutant and to inspect the effect of the mutations on the cytochrome’s heme core. The signals were assigned using the same methodology described for the wild-type protein.32 Briefly, the first step of the assignment encompasses the analysis of the 2D 1H-TOCSY spectra, in which the connectivities between the thioether methines (31H and 81H) and the thioether methyl groups (32CH3 and 82CH3) were identified. The next step includes the analysis of the NOE connectivities showed by the heme substituents in the 2D 1HNOESY spectrum. The meso protons present a characteristic pattern in this spectrum: (i) 15H meso protons are not connected to either methyl groups or thioether substituents, (ii) 20H protons are connected to two heme methyls (21CH3 and 181CH3) and (iii) both 5H and 10H protons present connectivities with a thioether methine (31H or 81H, respectively), a thioether methyl (32CH3 or 82CH3, respectively) and one heme methyl group (71CH3 or 121CH3, respectively). The latter ambiguity is solved by observing the connectivities between the heme methyls near the 20H protons (21CH3) with the closest thioether groups (31H and/or 32CH3), which are unequivocally assigned in the 2D 1H-TOCSY spectrum. This allows the connection between the heme edges containing 20H and 5H of each heme. The heme methyls 71CH3 are part of the heme 5H edges and also show NOE connectivities with thioether groups (81H and/or 82CH3), which are part of heme 10H edges. After the identification of each of the three heme edges, the 15H meso protons can be identified by observing its NOE connectivities with 121CH3 or 181CH3 methyls. An illustration with the expected connectivities for both types of NMR spectra is presented in the Supplementary Figure S1. With the exception of V13A, and in contrast to the wild-type cytochrome, all the mutants showed more than six connectivities in the thioether methine/thioether methyl proton region in the 2D 1H-TOCSY spectra (Fig. 2). The number of connectivities observed suggests that V13I, V13S and V13T mutants have two 7



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conformations displaying minor variations in the heme core in solution (hereafter conformations A and B). The presence of the two conformations was further confirmed in the 2D 1H-NOESY spectra of each mutant and is illustrated by the NOE connectivities between 5H and 10H meso protons and their respective thioether methine protons (Fig. 2). The assignment of the heme substituents in a multiheme cytochrome showing multiple conformations was achieved for the first time in the present work. The full list of assignments is provided in the Supplementary Table S1 and the comparison between the chemical shifts obtained for the mutated and wild-type cytochromes indicates that the overall heme core is not considerably affected despite displaying minor variations in two conformations (Fig. 3A). This is supported by the low RMSD values (Fig. 3A and Supplementary Table S1) and by the same set of NOE connectivities observed in 2D 1H-NOESY NMR spectra for both wild-type and mutants. Nonetheless, the RMSD values are slightly higher for heme III, which is not unexpected given its proximity to the mutated residue. The three heme groups are part of the protein’s hydrophobic core in which heme III is nearly perpendicular to both hemes I and IV (Fig. 1). Therefore, the nearest heme substituents of these two hemes are affected by the heme III ring-current effects and their chemical shifts are expected to be perturbed by minor structural rearrangement on this heme. For this reason, the RMSD values for hemes I and IV are comparable (Fig. 3A). Also, considerable chemical shift variations are observed for heme I substituents 82CH3, 81H and 10H even though this heme is located the farthest from V13 residue. As mentioned above, two conformational differences within the heme core are observable for V13I, V13S and V13T mutants (Fig. 2). The conformations showing the lowest and the highest chemical shift differences compared to PpcA were designated by ‘conformation A’ and ‘conformation B’, respectively. The data obtained shows that the perturbation on the heme core increases in the following order: V13I (total RMSD value of 0.06 ppm), V13S (0.11 ppm) and V13T (0.13 ppm), suggesting that the volume and polarity of the side chain of residue 13 is crucial to maintain a single conformation of the protein (Fig. 3B). In fact, in the V13A mutant, the small volume of the alanine residue (92 Å3)

34, 35

compared to valine (142 Å3) allows this

residue to fit in the hydrophobic core and only one conformation is observed in solution. This is corroborated by the crystal structure of V13A mutant (see the discussion below). On the other hand, the larger volume of isoleucine (169 Å3) perturbs the protein’s hydrophobic core yielding two conformations in solution. However, the perturbation of the heme core is even higher for the non-polar mutants. In this case, and despite their smaller volume compared to valine (99 and 122 Å3 for V13S and V13T, respectively) their polarity has a significant impact on the cytochrome’s heme core conformation. In both cases, two conformations were also observed in solution, which differ the most for the larger residue (V13T). Although V13S could not be crystallized, the crystal structure of V13T mutant showed two conformations for the threonine side chain (see discussion below). Remarkably, for the three mutants displaying two different conformations, the most affected heme substituents are conserved and are located in the edges pointing inside the protein hydrophobic core (see carbon atoms highlighted in red sticks in Fig. 3C). Compared to V13I and V13S, three additional heme protons (corresponding carbon atoms labeled with asterisks in Fig. 3C) were also affected in V13T which are in line with the largest perturbations caused by this replacement. 8


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Effect of the mutations on the heme oxidation profiles The dependence of the heme oxidation fraction with the redox potential for a monoheme cytochrome can easily be obtained, for example, from the variation of the area of the α-peak in a redox titration followed by UV-visible spectroscopy.36 This is attainable because only the reduced and oxidized stages can co-exist simultaneously in solution. However, in a triheme cytochrome, in addition to the fully reduced and oxidized stages, two intermediate oxidation stages can be defined: stage S1 and stage S2 (for a Review see37). Each of the four oxidation stages contains microstates with the same number of oxidized hemes and are interconnected by reversible one-electron transfer steps (Fig. 4). In the case of PpcA and the four V13 mutants studied in the present work, all the heme groups are axially coordinated by two histidine residues. Consequently, all have similar optical properties and for this reason the individual heme oxidation profiles cannot be distinguished from the analysis of redox titrations followed by UV-visible spectroscopy. We have shown previously for the wild-type cytochrome that the relative heme oxidation fractions can be monitored by 2D 1H-EXSY NMR experiments, a process that is facilitated by the low-spin character of the hemes.14 This was possible because the protein exhibited fast intramolecular (between the microstates within the same oxidation step) and slow intermolecular electron exchange rates (between microstates belonging to different oxidation stages) on the NMR time scale. In such exchange regime, it is possible to discriminate the heme proton substituent signals throughout the redox cycle of the protein using 2D 1H-EXSY NMR spectra. For such analysis, heme methyl substituents are the most appropriate ones because of their typical larger variation between the fully reduced and oxidized states (for a review see38). Just like the wild-type cytochrome, all the mutants exhibit fast intra- and slow intermolecular electron exchange on the NMR time scale. Regardless of the presence of different conformations in solution for three of the mutants, the chemical exchange connectivities could be monitored at pH 6. This is illustrated in the Supplementary Fig. S2. The observed chemical shift variations, in particular for V13S and V13T mutants, indicate that their heme oxidation fraction profiles are different compared to the wild-type cytochrome. Similarly to the wild-type cytochrome, 14 the heme oxidation fractions were determined from the chemical shifts of the heme methyls 121CH3I, 71CH3III, 121CH3IV observed in the distinct oxidation stages and are indicated in Table 2. The analysis of this Table confirms that also for the mutated cytochromes the extrinsic contributions to the observed chemical shifts are negligible. In fact, the sums of the oxidation fractions at each oxidation stage are close to integers, which confirms that the observed chemical shifts reflect the oxidation state of each particular heme. The oxidation patterns of the hemes for all proteins are represented in Figure 5A. In case of V13A and V13I the heme oxidation profiles are only slightly altered compared to the wild-type protein (cf. dashed and solid lines in Fig. 5A). As mentioned, the sums of the oxidation fractions at each oxidation stage are close to an integer and, therefore, the number of oxidized hemes is zero, one, two and three in oxidation stages 0, 1, 2 and 3, respectively (see Fig. 4). Thus, if one particular heme is more oxidized, the others are necessarily less. For this reason, the highest variation on the heme oxidation fraction may not correspond to the heme closest to the 9



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mutation site. This is illustrated for example for the mutant V13I (Fig. 5B). Although located further apart from the mutated residue, heme I shows slightly higher variation on the oxidation fraction values (approximately 10% in oxidation stages 1 and 2). This variation is caused by the cumulative increase in the oxidation fraction values of hemes III and IV, which are within van der Waals distance to residue V13. Despite these small variations, the heme oxidation profiles in the mutant are comparable to the wild-type, as well as their order of oxidation: I-IV-III. It is interesting to note that the replacement of V13 by a smaller residue (V13A) stabilizes the reduced state of heme III (small oxidation fraction values) whereas its replacement by a larger residue (V13I) yielded the opposite effect. In both cases, the oxidation fractions of heme IV are only marginally affected. In case of the polar replacements (V13S and V13T) a different scenario was observed. As discussed, the substitution of valine by serine and threonine residues caused a larger perturbation on the heme core compared to the non-polar replacements. This was further confirmed by the analysis of the heme oxidation profiles, which differ substantially from the wild-type cytochrome. In both mutants, the heme IV oxidation fractions increased compared to the wild-type, an effect that is even more prominent for V13T (Fig. 5B). The higher oxidation fraction values observed for heme IV indicate that the reduced state of the heme is more destabilized in the mutants, as expected by the introduction of a polar group in its vicinity. Heme III, also located in the vicinity of the mutated residue, showed a different behavior. In fact, its oxidation fractions are higher in V13S but lower in the V13T mutant, compared to the wild-type cytochrome (Fig. 5). These observations suggest that OH group of the side chain is positioned between heme III and IV in V13S, whereas in the case of V13T it is more oriented toward heme IV. To compensate the observed effect on hemes III and IV, the oxidation fractions of heme I are considerably smaller compared to the wild-type so that this heme is no longer the first one to oxidize (Fig. 5A and Table 2). Compared to the native protein, non-polar replacement mutants V13A and V13I show no differences in heme oxidation order, a fact not observed for V13S and V13T. In case of V13T, the heme oxidation profiles suggest a clear order of oxidation: IV-I-III, whereas in case of V13S the order is ambiguous in the sense that heme III is dominant in both stage 1 and stage 3 [III-(I, IV)-III]. Crystal Structures of V13A and V13T Structures of the two valine mutants, V13A and V13T, in the oxidized state are the same as the native PpcA. In case of V13A, RMSD for 71 alpha carbons is 0.13 Å, whereas for V13T RMSD for 71 alpha carbons is 0.11 Å. These rms deviations are smaller than the error in the coordinates. This confirms that, as in solution, the overall structure of the protein is conserved in the mutants. The Cα cartoon of the two mutants overlaid on the native PpcA is shown in Supplementary Figure S3 along with the three hemes. In V13T, the threonine side chain is disordered. The occupancy was not refined but based on visual inspection of the electron density the two orientations are given 50% occupancy each. This is in line with the results obtained in solution, where the NMR signals of V13T mutant suggested equal occupancy for both conformations (Fig. 2). Surprisingly, in the crystal structure there are larger deviations (two and three times the overall RMSD, respectively) in the alpha 10


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carbons of residues 13 and 14 compared to PpcA in case of V13A, but not in case of V13T. In case of V13T mutant, similarly higher deviations were observed in the alpha carbons of residues Asp26, Lys33 and Gly34. In addition, several side chains were different from each other in the two mutant structures, which could be related to differences caused by the crystals with the side chains frozen in one of the possible conformations. There were also some minor differences observed in the water positions.

Role of valine 13 in triheme cytochrome PpcA The side chain of valine 13 is well packed within the hydrophobic core formed by the protein and the three hemes in PpcA. Valine in this position is well conserved with a few exceptions where an isoleucine residue is substituted in the family of cytochromes c7 reflecting the necessary role for a hydrophobic side chain. The triheme cytochrome PpcA is quite temperature stable as can be seen in the circular dichroism (CD) heme signal (~ 404 nm) versus temperature plot (Supplementary Figure S4). The mid-point of transition (Tm) is not reached even at 90 °C. Similar data obtained for the hydrophobic mutants V13A and V13I, suggested that V13A may be slightly more stable than the native protein but V13I is slightly destabilized as expected from steric conflicts within the constraints of the protein-heme core and supported by the observation of two conformations in solution for this mutant. On the other hand, the CD data suggested a more prominent decrease in protein stability when a polar residue is introduced in the hydrophobic core of the protein in case of both V13T and V13S, with the latter mutant being the most affected. Replacement of valine 13 with a smaller residue (Ala) did not affect the structure or the heme oxidation order, whereas replacement by a larger non-polar residue (Ile) resulted in perturbation of the local structure in solution but did not affect the heme oxidation order. Replacement with a polar residue with similar (Thr) or smaller (Ser) volume resulted in significant variation in the heme oxidation order and structural perturbation in solution. From the analysis of V13 mutants in the present study, it is clear that valine at position 13 in PpcA is strategically placed in order to maintain the unique triheme core structure and the required heme oxidation order to enable the protein to participate in the e-/H+ coupling, explaining why this residue is highly conserved in the cytochrome c7 family of proteins. These results are in accordance with data obtained for other PpcA mutants. 22 Substitution of the highly conserved phenylalanine 15 residue, located in the hydrophobic core like valine 13, by tyrosine and tryptophan residues, resulted in sinal broadening of NMR spectra, suggesting the existence of different conformations in solution.

23

Thus, the data obtained in the present and previous works

show that the heme core is more desestabilized by the insertion of a polar or larger residue in the hydrophobic core. However, as revealed in the present study, the proteins are still robust enough to keep their ability to transfer and receive electrons. Thus, even in the presence of a slightly desestabilized heme core, the information obtained could be explored to design functional protein variants working at different redox potential ranges and enhance electron transfer directionality in respiratory chains.

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Conclusions In the present work, we studied the structural and functional role of a conserved residue located at the hydrophobic core of the triheme cytochrome PpcA from the bacterium Geobacter sulfurreducens. The selected residue, V13, is located within van der Waals distance to hemes III and IV. Two hydrophobic mutants (V13A and V13I) and two polar mutants (V13S and V13T) were functionally and structurally characterized using NMR, whereas two mutants that produced crystals, V13A and V13T were characterized by X-ray crystallography. The CD suggested that all mutants are temperature stable, just like the wild type protein. However, the hydrophobic mutants are slightly more stable than the polar mutants. The results showed that the nature of the side chain at position 13 has different impact on the redox properties of PpcA, as well as on the local structure of the heme core. While the replacement of residue V13 by alanine has no effect, the substitution of V13 by a larger hydrophobic residue (V13I) yielded two protein conformations with localized minor differences in the hemes. Nonetheless, in both cases, the oxidation profiles of the hemes are similar to the wild-type cytochrome with this order of oxidation: I-IV-III. On the other hand, for the polar mutants, V13S and V13T, the results showed that these residues have a more prominent effect both on the local structure of the protein and the heme core and on the redox properties of the heme groups. Two main features emerged compared to the wild-type: different conformations regardless of side chain size and an altered heme oxidation order resulting from the significant modification of the oxidation fraction values of the hemes. Previous studies have shown that the functional mechanism of PpcA relies on a fine-tuned regulation of the properties of the redox centres and the dominant microstates in solution.14, 20, 21, 23 The present study showed that the nature of residue at position 13 is crucial to maintain (i) a single conformation of the protein-heme hydrophobic core and (ii) the order of oxidation of the heme groups. Indeed, a large non-polar side chain disturbed the local structure of the heme core but maintained the redox properties of the hemes, whereas inclusion of a polar residue perturbed both the local structure and affected the order of oxidation of the hemes. Supporting Information Figure S1, Heme c nomenclature; Figure S2, Examples of 2D 1H-EXSY NMR spectra for wild-type protein and V13 mutants; Figure S3, The Cα tracings derived from the crystal structures of the V13A and V13T mutants overlaid on the native PpcA crystal structure; Figure S4, The normalized CD signal at the heme peak in the visible region for each protein is plotted against temperature; Table S1, Heme protons chemical shifts of wild-type protein and V13 mutants in the reduced state. ACKNOWLEDGMENTS This work was supported by Fundação para a Ciência e Tecnologia (FCT) through the following grants: SFRH/BD/86439/2012 (to APF), PTDC/BBB-BQB/3554/2014 and PTDC/BIA-BQM/31981/2017 (to CAS). This work was also supported by Unidade de Ciências Biomoleculares Aplicadas-UCIBIO which is financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under PT2020 12


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Partnership Agreement (POCI-01-0145-FEDER-007728). The NMR spectrometers are part of the National NMR Network (PTNMR) and are supported by Infrastructure Project Nº022161 (co-financed by FEDER through COMPETE 2020, POCI, and PORL and FCT through PIDDAC). PRP is partially supported by the division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy program under contract no. DE-AC02-06CH11357. REFERENCES (1) Weber, K. A.; Achenbach, L. A.; Coates, J. D. Microorganisms pumping iron: anaerobic microbial iron oxidation and reduction. Nat. Rev. Microbiol. 2006, 4, 752-764. (2) Morgado, L.; Fernandes, A. P.; Dantas, J. M.; Silva, M. A.; Salgueiro, C. A. On the road to improve the bioremediation and electricity-harvesting skills of Geobacter sulfurreducens: functional and structural characterization of multihaem cytochromes. Biochem. Soc. Trans. 2012, 40, 1295-1301. (3) Lovley, D. R. Electromicrobiology. Annu. Rev. Microbiol. 2012, 66, 391-409. (4) Wilkins, M. J.; Verberkmoes, N. C.; Williams, K. H.; Callister, S. J.; Mouser, P. J.; Elifantz, H.; N'Guessan A, L.; Thomas, B. C.; Nicora, C. D.; Shah, M. B., et al. Proteogenomic monitoring of Geobacter physiology during stimulated uranium bioremediation. Appl. Environ. Microbiol. 2009, 75, 6591-6599. (5) Speers, A. M.; Reguera, G. Electron donors supporting growth and electroactivity of Geobacter sulfurreducens anode biofilms. Appl. Environ. Microbiol. 2012, 78, 437-444. (6) Lovley, D. R.; Ueki, T.; Zhang, T.; Malvankar, N. S.; Shrestha, P. M.; Flanagan, K. A.; Aklujkar, M.; Butler, J. E.; Giloteaux, L.; Rotaru, A. E., et al. Geobacter: the microbe electric's physiology, ecology, and practical applications. Adv. Microb. Physiol. 2011, 59, 1-100. (7) Gorby, Y. A.; Yanina, S.; McLean, J. S.; Rosso, K. M.; Moyles, D.; Dohnalkova, A.; Beveridge, T. J.; Chang, I. S.; Kim, B. H.; Kim, K. S., et al. Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 11358-11363. (8) Mehta, T.; Coppi, M. V.; Childers, S. E.; Lovley, D. R. Outer membrane c-type cytochromes required for Fe(III) and Mn(IV) oxide reduction in Geobacter sulfurreducens. Appl. Environ. Microbiol. 2005, 71, 8634-8641. (9) Breuer, M.; Rosso, K. M.; Blumberger, J.; Butt, J. N. Multi-haem cytochromes in Shewanella oneidensis MR-1: structures, functions and opportunities. J. R. Soc. Interface 2015, 12, 20141117. (10) Santos, T. C.; Silva, M. A.; Morgado, L.; Dantas, J. M.; Salgueiro, C. A. Diving into the redox properties of Geobacter sulfurreducens cytochromes: a model for extracellular electron transfer. Dalton Trans. 2015, 44, 9335-9344. (11) Levar, C. E.; Chan, C. H.; Mehta-Kolte, M. G.; Bond, D. R. An inner membrane cytochrome required only for reduction of high redox potential extracellular electron acceptors. MBio 2014, 5, e02034. (12) Zacharoff, L.; Chan, C. H.; Bond, D. R. Reduction of low potential electron acceptors requires the CbcL inner membrane cytochrome of Geobacter sulfurreducens. Bioelectrochemistry 2016, 107, 7-13. (13) Malvankar, N. S.; Vargas, M.; Nevin, K.; Tremblay, P. L.; Evans-Lutterodt, K.; Nykypanchuk, D.; Martz, E.; Tuominen, M. T.; Lovley, D. R. Structural basis for metallic-like conductivity in microbial nanowires. MBio 2015, 6, e00084. (14) 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.

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(15) Ding, Y. H.; Hixson, K. K.; Aklujkar, M. A.; Lipton, M. S.; Smith, R. D.; Lovley, D. R.; Mester, T. Proteome of Geobacter sulfurreducens grown with Fe(III) oxide or Fe(III) citrate as the electron acceptor. Biochim. Biophys. Acta 2008, 1784, 1935-1941. (16) 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. (17) Morgado, L.; Bruix, M.; Pokkuluri, P. R.; Salgueiro, C. A.; Turner, D. L. Redox- and pH-linked conformational changes in triheme cytochrome PpcA from Geobacter sulfurreducens. Biochem. J. 2017, 474, 231-246. (18) Morgado, L.; Saraiva, I. H.; Louro, R. O.; Salgueiro, C. A. Orientation of the axial ligands and magnetic properties of the hemes in the triheme ferricytochrome PpcA from G. sulfurreducens determined by paramagnetic NMR. FEBS Lett. 2010, 584, 3442-3445. (19) Morgado, L.; Dantas, J. M.; Bruix, M.; Londer, Y. Y.; Salgueiro, C. A. Fine tuning of redox networks on multiheme cytochromes from Geobacter sulfurreducens drives physiological electron/proton energy transduction. Bioinorg. Chem. Appl. 2012, 2012, 298739. (20) Morgado, L.; Dantas, J. M.; Simoes, T.; Londer, Y. Y.; Pokkuluri, P. R.; Salgueiro, C. A. Role of Met58 in the regulation of electron/proton transfer in trihaem cytochrome PpcA from Geobacter sulfurreducens. Biosci. Rep. 2012, 33, 11-22. (21) Morgado, L.; Lourenço, S.; Londer, Y. Y.; Schiffer, M.; Pokkuluri, P. R.; Salgueiro, C. A. Dissecting the functional role of key residues in triheme cytochrome PpcA: a path to rational design of G. sulfurreducens strains with enhanced electron transfer capabilities. PLoS One 2014, 9, e105566. (22) Dantas, J. M.; Morgado, L.; Aklujkar, M.; Bruix, M.; Londer, Y. Y.; Schiffer, M.; Pokkuluri, P. R.; Salgueiro, C. A. Rational engineering of Geobacter sulfurreducens electron transfer components: a foundation for building improved Geobacter-based bioelectrochemical technologies. Front. Microbiol. 2015, 6, 752. (23) Dantas, J. M.; Morgado, L.; Londer, Y. Y.; Fernandes, A. P.; Louro, R. O.; Pokkuluri, P. R.; Schiffer, M.; Salgueiro, C. A. Pivotal role of the strictly conserved aromatic residue F15 in the cytochrome c7 family. J. Biol. Inorg. Chem. 2012, 17, 11-24. (24) 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. Acta 2013, 1827, 484-492. (25) Londer, Y. Y.; Pokkuluri, P. R.; Tiede, D. M.; Schiffer, M. Production and preliminary characterization of a recombinant triheme cytochrome c7 from Geobacter sulfurreducens in Escherichia coli. Biochim. Biophys. Acta 2002, 1554, 202-211. (26) Arslan, E.; Schulz, H.; Zufferey, R.; Kunzler, P.; Thony-Meyer, L. Overproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichia coli. Biochem. Biophys. Res. Commun. 1998, 251, 744-747. (27) Pokkuluri, P. R.; Londer, Y. Y.; Duke, N. E.; Long, W. C.; Schiffer, M. Family of cytochrome c7-type proteins from Geobacter sulfurreducens: structure of one cytochrome c7 at 1.45 Å resolution. Biochemistry 2004, 43, 849-859. (28) Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997, 276, 307-326. (29) Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros, P.; Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, M.; Pannu, N. S., et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D. Biol. Crystallogr. 1998, 54, 905-921. (30) Murshudov, G. N.; Vagin, A. A.; Lebedev, A.; Wilson, K. S.; Dodson, E. J. Efficient anisotropic refinement of macromolecular structures using FFT. Acta Crystallogr. D. Biol. Crystallogr. 1999, 55, 247-255. 14


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(31) Sack, J. S.; Quiocho, F. A. CHAIN: a crystallographic modeling program. Methods Enzymol. 1997, 277, 158-173. (32) Morgado, L.; Bruix, M.; Orshonsky, V.; Londer, Y. Y.; Duke, N. E.; Yang, X.; 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 2008, 1777, 1157-1165. (33) Goddard, T. D.; Kneller, D. G. (2007) Sparky 3.114. In. University of California, San Francisco, USA. (34) Richards, F. M. Areas, volumes, packing and protein structure. Annu. Rev. Biophys. Bioeng. 1977, 6, 151-176. (35) Baumann, G.; Frömmel, C.; Sander, C. Polarity as a criterion in protein design. Protein Eng. 1989, 2, 329-334. (36) Dantas, J. M.; Tomaz, D. M.; Morgado, L.; Salgueiro, C. A. Functional characterization of PccH, a key cytochrome for electron transfer from electrodes to the bacterium Geobacter sulfurreducens. FEBS Lett. 2013, 587, 2662-2668. (37) Turner, D. L.; Salgueiro, C. A.; Catarino, T.; Legall, J.; Xavier, A. V. NMR studies of cooperativity in the tetrahaem cytochrome c3 from Desulfovibrio vulgaris. Eur. J. Biochem. 1996, 241, 723-731. (38) Morgado, L.; Fernandes, A. P.; Londer, Y. Y.; Bruix, M.; Salgueiro, C. A. One simple step in the identification of the cofactors signals, one giant leap for the solution structure determination of multiheme proteins. Biochem. Biophys. Res. Commun. 2010, 393, 466-470. (39) Turner, D. L.; Costa, H. S.; Coutinho, I. B.; Legall, J.; Xavier, A. V. Assignment of the ligand geometry and redox potentials of the trihaem ferricytochrome c3 from Desulfuromonas acetoxidans. Eur. J. Biochem. 1997, 243, 474-481. (40) Fernandes, T. M.; Morgado, L.; Salgueiro, C. A. Thermodynamic and functional characterization of the periplasmic triheme cytochrome PpcA from Geobacter metallireducens. Biochem. J. 2018, 475, 28612875.

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FIGURE LEGENDS Fig. 1. Spatial location of the residue V13 in the PpcA solution structure (PDB code, 2LDO16). The PpcA polypeptide chain (blue) is shown as Cα ribbon and heme groups (gray). The side chain of V13 (red) is represented as stick drawings. The hemes are numbered I, III and IV, a designation that derives from the superimposition of the hemes in cytochromes c7 with those of the structurally homologous tetraheme cytochromes c3.39 Fig. 2. Selected regions of 2D 1H-TOCSY and 2D 1H-NOESY NMR spectra to illustrate the presence of different conformations in solution. The connectivities between the thioether methines (31H or 81H) and the thioether methyl groups (32CH3 and 82CH3) are illustrated in the 2D 1H-TOCSY spectra (left panels). The NOE connectivities between 5H and 10H meso protons and their respective thioether methine protons are illustrated in the 2D 1H-NOESY spectra (right panels). Black and blue labels represent the signals of conformations A and B, respectively. Fig. 3. Impact of the mutations on the heme core architecture. (A) Comparison of the heme proton chemical shifts of the PpcA valine mutants (MUT) and those of PpcA (WT). Green, orange, and blue symbols correspond to hemes I, III, and IV, respectively. The RMSD values calculated from the chemical shifts measured for the wild-type and mutants are: (i) V13A: 0.01, 0.05 and 0.01 ppm for hemes I, III and IV, respectively; (ii) V13I: 0.01(0.05), 0.05(0.08) and 0.02(0.03); (iii) V13S: 0.06(0.13), 0.07(0.13) and 0.05(0.08); (iv) V13T: 0.01(0.15), 0.03(0.15) and 0.03(0.09). The values given in parenthesis correspond to the conformation B. The solid line has a unit slope. (B) Variation of the heme proton chemical shifts in conformations A and B (MUT(A) - MUT(B)) for V13I, V13S and V13T. The green, orange, and blue bars correspond to hemes I, III, and IV, respectively. (C) Heme core of PpcA (PDB code, 2LDO16) highlighting the most affected signals in the two conformations for V13I, V13S and V13T. The carbon atoms bonded to the most affected protons in the three mutants are colored red. Blue asterisks indicate carbon atoms bonded to additional protons that are also affected in the V13T mutant. Fig. 4. Electronic distribution network in a triheme cytochrome. In each microstate the heme groups are colored green (heme I), orange (heme III) and blue (heme IV), which can be either reduced (filled symbols) or oxidized (open symbols). The microstates are grouped, according to the number of oxidized hemes, in four oxidation stages connected by three one-electron redox steps (for a review, see40). P0 represents the reduced microstate. Pijk corresponds to the microstates with heme(s) i, j, and k oxidized. Fig. 5. Impact of the mutations on the heme oxidation order. (A) Oxidation fraction of V13 mutants (solid symbols and lines) and PpcA (open symbols and dashed lines). (B) Variation of the oxidation fractions (xi) relative to the wild-type (xi(MUT) - xi(WT)). Data for hemes I, III and IV are colored in green, orange and 16 ACS Paragon Plus Environment

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blue, respectively. The heme oxidation fractions were calculated according to equation xi = (i-0)/(3-0), where i, 0, and 3 are the observed chemical shifts of each methyl in stages i, 0, and 3, respectively (see Fig. 4). The chemical shifts are listed in the Table 2.

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FIGURE 1


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FIGURE 2



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FIGURE 3


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FIGURE 4



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FIGURE 5


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Table 1. Crystallographic parameters and refinement statistics. V13A

V13T

a= b=32.2 Å, c=178.8 Å

a= b=32.4 Å, c=178.4 Å

P4322

P4322

1

1

VM (Å3/Da) (% solvent)

3.00 (59)

3.04 (60)

Wavelength (Å)

1.03320

1.03320

Resolutiona (Å)

90-1.9 (1.97-1.90)

Crystal & data parameters Unit cell dimensions Space group #mol/AU

R-mergea

90-1.95 (2.02-1.95)

0.040 (0.087)

0.072 (0.102)

5.9 (2.4)

5.8 (2.5)

94.5 (73.8)

91.4 (61.9)

58 (16)

31 (7.1)

Refmac5

Refmac5

Resolution range (Å)

30-1.9

30-1.95

Number of reflections

6920

6269

R-factor

0.188

0.185

R-freeb

0.229

0.233

Redundancy Completeness (%) Mean I/σ(I) a Refinement Program used

Number of non-hydrogen atoms (mean B-factor, Å2) Protein

538 (18.9)

539 (20.6)

Heme

129 (13.7)

129 (16.2)

Deoxycholate

28 (16.3)

28 (16.2)

Solvente

88 (31.3)

85 (33.9)

0.016

0.016

1.5

1.6

4HBF

4HC3

RMSD Bonds (Å) Bond angles (°) PDB code a

Values in parentheses are for the highest resolution shell. b Test set consisted of 10% reflections.



Page 24 of 25

Table 2. Redox-dependence of the heme methyl proton chemical shifts and heme oxidation fractions for PpcA and V13 mutants (pH 6 and 288 K). As for the wild-type, the heme methyls 121CH3I, 71CH3III and 121CH3IV were used to monitor each heme oxidation through the four oxidation stages. The heme oxidation fractions, xi, in each stage of oxidation, were calculated according to the equation xi = (i-0)/(3-0), where i, 0, and 3 are the observed chemical shifts of the heme methyl in stage i, 0 (fully reduced) and 3 (fully oxidized), respectively. The values for PpcA were previously determined14 and are listed for comparison. Protein

Oxidation stage

V13A

V13I

V13S

V13T

PpcA

Chemical shift (ppm)

 xi

xi

I

III

IV

I

III

IV

0

2.55

4.14

3.94

0

0

0

0

1

13.12

7.34

6.43

0.56

0.23

0.16

0.95

2

19.87

10.72

12.56

0.92

0.47

0.56

1.96

3

21.34

18.01

19.35

1

1

1

3

0

2.55

4.14

3.95

0

0

0

0

1

10.85

9.1

6.25

0.43

0.35

0.17

0.95

2

18.39

12.03

11.62

0.82

0.55

0.58

1.95

3

21.93

18.46

17.21

1

1

1

3

0

2.53

4.14

4.02

0

0

0

0

1

6.92

9.86

8.30

0.23

0.42

0.33

0.97

2

15.06

11.93

13.76

0.65

0.57

0.75

1.96

3

21.89

17.92

17.05

1

1

1

3

0

2.47

4.11

3.99

0

0

0

0

1

7.66

6.41

10.46

0.27

0.16

0.56

0.99

2

18.06

7.90

14.04

0.82

0.26

0.87

1.95

3

21.50

18.49

15.53

1

1

1

3

0

2.55

4.14

3.95

0

0

0

0

1

12.70

8.13

5.92

0.53

0.28

0.14

0.96

2

19.78

11.61

11.19

0.90

0.52

0.53

1.95

3

21.62

18.42

17.69

1

1

1

3


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Structural and Functional Relevance of the Conserved Residue V13 in

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