Unveiling the Structural Basis That Regulates the Energy Transduction

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Unveil the Structural Basis that Regulates the Energy Transduction Properties within a Family of Triheme Cytochromes from Geobacter sulfurreducens Joana M Dantas, Telma Simões, Leonor Morgado, Clara Caciones, Ana P. Fernandes, Marta A Silva, Marta Bruix, Phani Raj Pokkuluri, and Carlos A. Salgueiro J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b07059 • Publication Date (Web): 07 Sep 2016 Downloaded from http://pubs.acs.org on September 13, 2016

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The Journal of Physical Chemistry

Unveil the Structural Basis that Regulates the Energy Transduction Properties within a Family of Triheme Cytochromes from Geobacter sulfurreducens

Joana M. Dantas,a Telma Simões,a Leonor Morgado,a,# Clara Caciones,a Ana P. Fernandes,a Marta A. Silva,a Marta Bruix,b P. Raj Pokkuluri,c and Carlos A. Salgueiroa,*

a

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

NOVA de Lisboa, Campus Caparica, 2829-516 Caparica, Portugal b

Departamento de Química Física Biológica, Instituto de Química Física Rocasolano, CSIC, Madrid,

Spain c

Biosciences Division, Argonne National Laboratory, Lemont, Illinois 60439, USA

# Present Address: Biozentrum, University of Basel, Basel, Switzerland *

Corresponding author: [email protected]

Telephone: (+351) 212 948 300

1 ACS Paragon Plus Environment


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ABSTRACT A family of triheme cytochromes from Geobacter sulfurreducens plays an important role in extracellular electron transfer. In addition to their role in electron transfer pathways, two members of this family (PpcA and PpcD) were also found to be able to couple e-/H+ transfer through the redoxBohr effect observed in the physiological pH range, a feature not observed for cytochromes PpcB and PpcE. As part of understanding the molecular control of the redox-Bohr effect in this family of cytochromes, which is highly homologous both in amino acid sequence and structures, it was observed that residue 6 is a conserved leucine in PcpA and PpcD, whereas in the other two characterized members (PpcB and PpcE) the equivalent residue is a phenylalanine. To determine the role of this residue located close to the redox-Bohr center, we replaced Leu6 in PpcA with Phe and determined the redox properties of the mutant, as well as its solution structure in the fully reduced state. In contrast with the native, the mutant PpcAL6F is not able to couple e-/H+ pathway. We carried out the reverse mutation in PpcB and PpcE (i.e., replace Phe6 in these two proteins by leucine) and show that the mutant proteins showed an increased redox-Bohr effect. The results clearly establish the role of residue 6 in the control of the redox-Bohr effect in this family of cytochromes, a feature that can enable rational design of G. sulfurreducens strains carrying mutant cytochromes with optimal redox-Bohr effect as suitable for various biotechnological applications.


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INTRODUCTION Geobacter species represent a rare example of microorganisms that are abundant in a diversity of natural environments.1 They can use different electron donors and acceptors that include insoluble metals and electrode surfaces.2 For these reasons these microorganisms were selected as targets for bioremediation of contaminated sediments in natural environments3, microbial electrochemical devices.5,

6

4

and as current producers in

However, when extracellular electron acceptors are used by

Geobacter cells, the biomass production decreases considerably.7,

8

Therefore, understanding the

molecular and structural basis of the mechanisms that contribute to energy transduction in these bacteria would provide important tools to improve the bacteria’s biomass yields and contribute to optimize the Geobacter-based practical applications. Periplasmic cytochromes play an important role in extracellular electron transfer (EET) by bridging the electron transfer between the inner membrane associated components and those located at the outer membrane.9 A family of five periplasmic triheme cytochromes (designated PpcA-E) was identified in Geobacter sulfurreducens and has been studied in detail by biochemical and biophysical methods.9-22 These cytochromes share a considerable important degree of amino acid sequence identity (Figure 1A). The thermodynamic parameters obtained for the aforementioned cytochromes, except for PpcC, which presented multiple conformations in solution that impaired the monitorization of its heme oxidation profiles,23 showed that the proteins present different working functional ranges.13 All the heme reduction potentials are modulated by the oxidation state of neighbouring hemes (redox interactions) and by the solution pH (redox-Bohr interactions) that provide PpcA and PpcD the necessary properties to couple e-/H+ transfer in the physiological pH range (via redox-linked acid-base equilibria designated redox-Bohr effect).13, 18 Such properties were not observed for PpcB and PpcE.13 The four triheme cytochromes showed the highest redox-Bohr interaction with heme IV suggesting that the redox-Bohr center is located in the vicinity of this heme13. In fact, we previously identified propionate 13 of heme IV (P13IV) as the protonatable center 3 ACS Paragon Plus Environment


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that is responsible for the redox-Bohr effect in PpcA and PpcB,11, 12 so it is conceivable that P13IV is also the redox-Bohr center in PpcD and PpcE. The pKa values of the redox-Bohr center in the fully reduced and fully oxidized state were determined for the four proteins and the difference in these values (∆pKa = pKared − pKaox) correlates with the magnitude of the observed redox-Bohr effect.13 The larger redox-Bohr effect (∆pKa) was observed for PpcA and PpcD, 2.1 and 1.8 pH units, respectively. The ∆pKa value is lower for PpcB and considerably smaller for PpcE (1.1 and 0.3 pH units, respectively). The high level of structural homology amongst G. sulfurreducens triheme cytochromes12,

19

suggests that the properties of their redox-Bohr centers are fine-tuned by the chemical environment of P13IV and, thus, provide an excellent case to investigate the molecular basis of the redox-Bohr effect that allows these proteins to regulate the proton-uptake/release processes occurring in the physiological pH range for cellular growth. Residue 6 is one of the residues located close to the redox-Bohr center (Figure 1B-D) in the structures of these cytochromes. It is striking that residue 6 is a leucine in both PpcA and PpcD, both the proteins exhibiting significant redox-Bohr effect, whereas the same residue is a phenylalanine in PpcB and PpcE that do not show significant redox-Bohr effect. In the present work, we aimed to understand the role of the amino acid present at residue 6 in the modulation of the redox-Bohr effect in the G. sulfurreducens periplasmic triheme cytochromes. Therefore, residue Leu6 was replaced by phenylalanine in PpcA (PpcAL6F). In addition, Phe6 was replaced by leucine in PpcB (PpcBF6L) and PpcE (PpcEF6L) and their redox behaviors were monitored. The thermodynamic properties and the functional mechanism of PpcAL6F were determined using visible and NMR spectroscopy.

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N-labeled PpcAL6F was also produced and the

solution structure of PpcAL6F in a fully reduced state was determined. In addition, the pH-linked conformational changes associated with the protonation/deprotonation of the redox-Bohr center in PpcAL6F were determined. The comparison of the redox and structural properties of this cytochrome 4 ACS Paragon Plus Environment

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with those, previously determined for the wild-type protein13,

24

revealed the crucial role of the

targeted residue in the regulation of the redox-Bohr effect in the G. sulfurreducens triheme cytochromes.

MATERIALS AND METHODS Site Directed Mutagenesis, Bacterial Growth and Purification All the mutants characterized in the present work (PpcAL6F, PpcBF6L or PpcEF6L) were produced using the QuikChange Site-Directed Mutagenesis Kit (NZYTech), as previously described for other unrelated PpcA mutants.14-17 Uniformly

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N labeled PpcAL6F and unlabeled PpcAL6F,

PpcBF6L or PpcEF6L were expressed in Escherichia coli strain BL21(DE3) and purified, as previously described for the wild-type proteins.25 NMR Studies on PpcAL6F Preparation of NMR Samples and NMR Experiments NMR samples, experimental conditions and NMR spectra matched exactly with those used in the thermodynamic and structural characterization of the wild-type protein.12, 13 For the thermodynamic studies, which were carried out at intermediate stages of oxidation, PpcAL6F mutant samples were prepared in 80 mM phosphate buffer with NaCl (250 mM final ionic strength) in 2H2O. For NMR experiments underlying the structural studies, PpcAL6F samples of approximately 1 mM were prepared in 45 mM phosphate buffer with NaCl (100 mM final ionic strength) pH 7.1. All the NMR experiments were acquired in Bruker Avance III 600 or 800 spectrometers equipped with triple-resonance cryoprobes. The latter spectrometer was used to obtain data to assist the solution structure determination of PpcAL6F resulting in comparable number of constraints for the mutant and the wild-type cytochromes (see below). The 1H and 15N chemical shifts were calibrated as previously described for the wild-type protein.24 For thermodynamic studies, the stepwise



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oxidation of the individual hemes in PpcAL6F was monitored by 2D 1H- EXSY spectra, as described for the wild-type protein.13 For the solution structure determination, the following set of experiments was acquired at 298 K: (i)

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N labeled sample, 2D 1H,15N- HSQC; (ii) unlabeled sample, 2D 1H- COSY, 2D 1H-TOCSY

with 60 ms mixing time and 2D 1H-NOESY with 50 ms mixing time. The effect of pH titration on the PpcAL6F chemical shifts was determined by the analysis of a series of 2D 1H,15N-HSQC spectra acquired in the pH range 5.5 to 9.5, as described for the wild-type cytochrome.24 Assignment of the NMR Signals The specific assignment of the PpcAL6F heme and polypeptide chain signals were obtained using the methodologies previously described for the wild-type protein.11, 13, 24 Data was deposited in the BioMagResBank (http://www.bmrb.wisc.edu) under BMRB accession number 25874. Structure Calculation and Analysis The structure of PpcAL6F was determined by NMR according to the methodology used for the wild-type cytochrome 24 and the data has been deposited in the Protein Data Bank with accession code 2N91. Redox Titrations of the Mutated Cytochromes Followed by Visible Spectroscopy The anaerobic potentiometric redox titrations followed by visible spectroscopy of PpcAL6F, PpcBF6L and PpcEF6L mutants were carried out at pH 7.0 and 8.0 using experimental conditions matching exactly with those used for the wild-type proteins.11, 13 The experiments were reproducible and all the reduction potentials referred in the present work are relative to normal hydrogen electrode, NHE). Thermodynamic Model The theoretical framework of the thermodynamic model used to describe in detailed thermodynamic properties of the redox centers in multiheme proteins, in general, and in triheme 6 ACS Paragon Plus Environment

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cytochromes in particular was previously described.13,

18, 26

The illustration of the different redox

stages in a triheme cytochrome and distribution of the microstates within each oxidation stage is provided in the Supplementary Figure S1 (for a review see26). The thermodynamic characterization of PpcAL6F mutant was carried out as described for the wild-type cytochrome13 using the same set of heme methyls (121CH3I, 71CH3III, 121CH3IV in each oxidation stage and in the pH range 5.5 to 9.5.

RESULTS AND DISCUSSION The detailed thermodynamic characterization of triheme cytochromes PpcA, PpcB, PpcD and PpcE from G. sulfurreducens showed that the redox potentials of the hemes are modulated by redox and redox-Bohr interactions.13 It was also showed that the ability to couple e-/H+ transfer in the physiological pH range for these cytochromes is distinct. Such mechanisms were observed for PpcA and PpcD but not for PpcB or PpcE. We noted that residue 6 located close to the redox-Bohr center is a leucine in both PpcA and PpcD, whereas the same residue is a phenylalanine in both PpcB and PpcE. In the present work, we analyzed the role of residue 6 in the modulation of the redox-Bohr center properties by replacing Leu6 with a phenylalanine residue in PpcA (PpcAL6F) followed by the determination of its detailed thermodynamic properties and solution structure. Impact of the Mutations on the Global Fold and Heme Core 1

H,15N-HSQC NMR experiments were used to fingerprint the overall structure of PpcAL6F and to

evaluate the impact of the mutation on the protein conformation. The comparable dispersion of the amide signals in the 1H,15N-HSQC NMR spectra obtained for PpcAL6F and wild-type cytochrome shows that most of the signals are overlapping, indicating that the native fold of the polypeptide chain is conserved (Figure 2). The most affected NH signal corresponds to the amide group of Phe6, the mutated residue. The other affected signals correspond to neighboring residues located in the polypeptide segment: Ile4-Ala8 and residues Asn10 and Phe15. These two latter residues are located across from residues Ala8 and Phe6, respectively. Furthermore, the amide signal of Ile38, His47 and 7 ACS Paragon Plus Environment


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Lys70 are slightly displaced from its position in the native protein. All these residues are also located in the vicinity of heme IV or near residues that were also affected by the mutation. In fact, Ile38 is part of the protein hydrophobic core and is located in the proximity of aromatic residues Phe6 and Phe15; His47 is one of the axial ligands of heme IV and Lys70 is placed at the C-terminus in the proximity of residue Asn10. Thus, it can be concluded that the replacement of Leu6 residue led to small conformational rearrangement of the neighboring residues without affecting the global fold of the protein. The impact of the mutation on the heme core architecture of PpcAL6F mutant was also probed by 2D 1H-NMR. The heme proton resonances were assigned as previously described 11 and are listed in Supplementary Table S1. The rmsd values between the chemical shifts measured for PpcAL6F and those of PpcA are low (0.09; 0.05; 0.13 ppm for hemes I, III and IV, respectively), though slightly higher for heme I and IV signals, as expected from the location of residue 6 between these two heme groups. The good correlation obtained in chemical shifts of heme protons in both proteins indicates that the neighborhoods of each heme were unaffected by the mutation. The NOE connectivities between the heme groups were also analyzed, and showed the same set of connectivities for both cytochromes. Taken together, the results obtained confirm that the heme core arrangement is conserved between the two cytochromes. Redox Characterization of Heme Groups and Redox-Bohr Center After confirming that the global fold and the heme core arrangement are conserved we probed the effect of replacing the Leu6 residue by phenylalanine on the functional properties of the cytochrome. In order to achieve this, the detailed thermodynamic characterization of PpcAL6F mutant was carried out in the same experimental framework used for the wild-type protein.13 To illustrate the stepwise oxidation of PpcAL6F mutant, 2D 1H-EXSY NMR spectra obtained at pH 6.0 are presented in Supplementary Figure S2. The same type of NMR spectra were collected at different pH values and the chemical shifts of heme methyls were measured for different oxidation stages (Figure 3). 8 ACS Paragon Plus Environment

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Compared to the wild-type the heme methyl 121CH3IV, which is closely located to the mutation site is the most affected one (cf. red and black lines in Figure 3). In order to quantify the effect of the mutation on the redox properties of the heme groups, the thermodynamic parameters were determined from the fitting of the pH dependence of the heme methyl chemical shifts and visible redox titrations, as described for the wild-type cytochrome.13 The thermodynamic parameters obtained for PpcAL6F are indicated in Table 1. Compared to the wildtype protein, the reduction potential values of the hemes are slightly more negative for the fully reduced and protonated protein (Table 1). In addition, the heme-heme redox interactions are also smaller and similarly affected. The higher redox-Bohr interaction observed with heme IV in the mutant, indicates that the redox-Bohr center remains associated with heme IV, as observed in the native protein.11-13 The redox-Bohr interactions are clearly more affected, particularly for heme IV, indicating that residue at position 6 is implicated in the modulation of the properties of the redoxBohr center (P13IV). Impact of the Mutation on the Order of Oxidation of the Hemes at Physiological pH The relative order of oxidation of the heme groups in the fully protonated and reduced proteins can be obtained from the values of the microscopic reduction potentials and heme-heme redox interactions (Table 1). For both proteins the order of oxidation of the hemes is I-III-IV. However, in order to proper analyse the protein’s redox function, the heme reduction potentials, and therefore, the order of oxidation of the hemes should be determined at physiological pH for G. sulfurreducens growth. To evaluate the effect of the mutation on the heme midpoint reduction potentials (eapp) at pH 7.5, the oxidation curves of the individual hemes were calculated from the thermodynamic parameters listed in Table 1 (Figure 4A). As a consequence of the deprotonation of the redox-Bohr center at physiological pH, the affinity of each redox centre for the electron is also modulated by the redox-Bohr interactions such that their apparent midpoint reduction potentials eapp differ from those in the fully reduced protein (Figure 4A). The shape of the curves in the mutant, as well as their 9 ACS Paragon Plus Environment


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separation, is also different compared to the wild-type protein, which reflects the differences in their redox parameters and particularly, in the heme reduction potentials (cf. solid and dashed lines in Figure 4A). The analysis of the individual oxidation profiles of PpcAL6F clear indicates that the oxidation profile of heme IV located in the proximity of the mutated residue is the most affected one. Compared to the wild-type, the eapp values are smaller (24, 19 and 32 mV for hemes I, III and IV, respectively). Despite the larger variation on the eapp value of heme IV (32 mV) the order of oxidation of the hemes (I-IV-III) is preserved in both proteins. The lowering of the heme IV eapp value may be rationalized by the inclusion of partial negative charge of the phenylalanine ring at position 6, which is expected to stabilize the oxidized form of this heme, and/or by the increase in its solvent exposure (see below). Role of Leu6 on the Functional Mechanism of Triheme Cytochrome Family As shown above, the oxidation profile of the redox centers is affected by the replacement of leucine by phenylalanine residue at position 6. In order to evaluate the effect of the mutation on the PpcA functional mechanism, the relative contribution of each 16 microstates (see Supplementary Figure S1) along the redox cycle of the protein was determined (Figure 4B). Such study was previously done for the wild-type protein13 and an e-/H+ transfer step is clearly observed between oxidation stages 1 and 2 involving the microstates P1H and P14 (Fig. 4B). From the analysis of Figure 4B, it is clear that the dominant microstates are different in the PpcAL6F mutant. Based on the preferential route for electrons observed for PpcA, and since heme I is oxidized in the two functional microstates, it was proposed that the heme I region interacts with its electron acceptor whereas that of heme IV, which alters its oxidation state between the one-and two-electron oxidized protein, is the interaction region with the electron donor.13,14 The preferred pathway observed for the wild-type protein is clearly disrupted in PpcAL6F, which is illustrated by the fact that the dominant microstates are all deprotonated (cf. dashed and solid lines in Figure 4B). This is a consequence of the significant decrease of the redox-Bohr center pKa value in the mutant (see Table 2). Therefore, while in the 10 ACS Paragon Plus Environment

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wild-type the larger redox-Bohr effect (∆pKa = pKared - pKaox) warrants that deprotonation events can occur in the entire physiological pH range, this is not the case for PpcAL6F. Remarkably, for cytochromes PpcB and PpcE the total redox-Bohr effect is smaller compared to PpcA (1.1 and 0.3 pH units for PpcB and PpcE, respectively) and no preferential pathway for e-/H+ transfer was observed for both proteins.13 The magnitude of the redox-Bohr effect in the physiological pH range can be simply evaluated by the separation of the apparent macroscopic redox potential from the potentiometric redox titrations obtained at different pH values. To illustrate this, the redox titrations previously obtained for PpcA, PpcB, PpcD, PpcE are depicted in Figure 5. From this analysis it is clear that the curves for cytochromes that can couple e-/H+ transfer (PpcA and PpcD) are more separated. In order to further test this in the present work we replaced Phe6 by leucine in PpcB (PpcBF6L) and PpcE (PpcEF6L) and the effect of such mutation on the redox titrations curves was evaluated (Figure 5). Compared to the respective wild-type proteins, the separation of the redox titration curves increased upon replacement of the aromatic residue at position 6 by a leucine in PpcB and PpcE. This indicates that an aromatic residue located in the vicinity of the redox-Bohr center decreases the total redox-Bohr effect and prevents the proteins to perform e-/H+ transfer. Taken together these observations, it is clear that residue 6 plays a crucial role in the functional mechanism of each cytochrome by modulating the pKa value of the redox-Bohr center and, concomitantly, by controlling the microscopic redox states that can be accessed during the redox cycle. The ability of the proteins to couple e-/H+ transfer is favored by a larger redox-Bohr effect in the physiological pH region and might be important to the periplasmic proton electrochemical gradient. To structurally rationalize the above described observations, we have also determined the solution structure of PpcAL6F in the fully reduced state and mapped the pH-linked conformational changes. Solution Structure Determination of PpcAL6F



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A summary of the sequential connectivities between the backbone protons is indicated in Supplementary Figure S3. An average of 33 NOE restraints per amino acid residue (14 lov and 19 upv) and 160 per heme residue (68 lov and 92 upv) were used for the final calculation (Figure 6A), following the same methodology used to determine the solution structure of the wild-type cytochrome.24 The final family consists of 20 structures (Figure 6B) with the lowest target function values (from 1.65 to 1.95 Å2 with an average value 1.81 Å2). The structures superimpose with an average pairwise backbone (N-Cα-C’) rmsd of 0.33 Å and a heavy atom rmsd of 0.96 Å (Supplementary Figure S3). The statistics for this family of structures are shown in Table 3. The solution structure of PpcAL6F consists of a two-strand antiparallel β-sheet at the N-terminus formed by Val5-Phe6 and Val13-Lys14, followed by three α-helices between residues His17-Ala23, Lys43-His47 and Gly53-Glu56 (Figure 6C). Structural Comparison of PpcAL6F and PpcA A detailed comparison of the best superposition of conformers describing the structure of PpcAL6F and native cytochromes in solution was performed indicating that the overall fold is conserved in both cytochromes (Figure 7). The parameters describing the heme geometry of PpcAL6F in solution are presented in Table 4 and shows that the heme core is also conserved. The iron-iron distances and the angles between the heme planes differ by less than 6 and 10%, respectively. The heme exposures are 242.3 Å2 (231.7 Å2 for wild-type), 213.6 Å2 (215.8 Å2) and 209.9 Å2 (171.3 Å2) for hemes I, III and IV, respectively. Compared with the wild-type protein, hemes I and IV showed a slightly increase in the solvent exposure, which explains their more negative reduction potential values. In particular, heme IV whose potential is most affected by the mutation (-158 mV versus -126 mV in the wild-type cytochrome, see Figure 4A) shows the largest increase in the solvent exposure. The reduction potential of heme III is also lower in the mutant though its solvent exposure is similar in both proteins. Such behavior is explained by the decrease in the heme redox interaction values with heme III in the mutant. In fact, the sum of the redox 12 ACS Paragon Plus Environment

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interactions involving heme III is 55 mV in the mutant compared to 68 mV in the native protein (Table 1). Since hemes I and IV oxidize prior to heme III, as their oxidation progress the affinity of heme III for electrons increases, as a consequence of the positive redox interactions between the heme groups. Therefore, in both native and mutant protein, heme III is the last heme to oxidize. However, because the smaller value of the redox interactions with heme III the midpoint reduction potential of this heme is smaller in the mutant cytochrome (see Figure 4A). Structural Mapping of the Redox-Bohr Center

In order to rationalize the observed changes in the properties of the redox-Bohr center we further study the effect of its protonation/deprotonation on the PpcAL6F structure. The higher value observed for the redox-Bohr interaction with heme IV (Table 2) suggests that the redox-Bohr center is also placed in the vicinity of heme IV in the mutant. The pH titration of PpcAL6F was carried out as previously described for the wild-type cytochrome24 and all backbone amide signals (except for residues 1 and 2) were assigned. The analysis of the chemical shift of these signals showed that the backbone amide signals of Lys7, Ala8, Asn10, Ile38 and the side chain of Asn10 are the most affected (Figure 8). The same set of signals were also the most affected ones in the native protein24, however the magnitude of the chemical shift perturbation differs (Figure 8). The effect of the pH on the NH signal from Ile38 was previously described in the wild-type cytochrome and is not related with the protonation/deprotonation of the redox-Bohr center.24 On the other hand, residues Lys7, Ala8 and Asn10 are placed in the vicinity of heme IV (Figure 8A) and are likely to be affected by the protonation/deprotonation events of the redox-Bohr center. The pH titration of the most affected signals is indicated in Figure 8B. As for the wild-type protein, the NH signals of Lys7, Ala8 and Asn10 have circumneutral pKa values of 7.0, 7.1 and 6.9, respectively. The pKa values of the most affected NH signals in the vicinity of heme IV (Lys7, Ala8,



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Asn10) are on average 0.5 pH units lower in the mutant, which correlates with the observed decrease in the pKred values of the redox-Bohr center (see Table 2). The structural features underlying the origin of the redox-Bohr effect observed in PpcA were previously identified.12 The structure of the wild-type protein showed that carbonyl oxygen of Lys7 is within hydrogen bonding distance from P13IV and it was proposed that the deprotonation of P13IV disrupts the hydrogen bond with Lys7, which explain the observed pH-linked conformational changes in the region of heme IV. Interestingly, the solution structure of PpcAL6F showed that the solvent exposure of heme IV increases, as well as the exposure of the propionate P13IV compared to the native protein. However, this propionate group showed structural variation in the family of structures. Analysis of the distance between the Lys7 O7 and heme IV O1D in each structure of the family clearly showed that it is smaller in the native cytochrome, with an average value of 2.8 Å compared to an average value of 3.3 Å in the mutant. Similarly, the surface exposure of this propionate group is higher in the mutant compared to the native protein (average accessible surface area for the carboxyl oxygen atoms of P13IV in the family of the structures for mutant is 24 Å2 in contrast with an average value of 16 Å2 for that of the native). The increase in the heme IV solvent exposure correlates with the decrease of the pKa value of the redox-Bohr center in the mutant, while the average higher distance observed between Lys7 O7 and P13IV explains the smaller chemical shift perturbation in the most affected NH signals in the vicinity of heme IV (cf. open and close symbols in Figure 8A), confirming that the properties of the redoxBohr center in triheme cytochromes from G. sulfurreducens are highly controlled by the nature of the residue in position 6. Aromatic Rings and the Redox-Bohr Behavior of the Triheme Cytochrome Family

Another interesting observation is that the two cytochromes (PpcA and PpcD) that can couple e/H+ transfer within the physiological pH range for Geobacter growth have two internal aromatic side chains (other than heme binding histidine side chains; see Table 5) whereas the other two 14 ACS Paragon Plus Environment

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cytochromes of the family (PpcB and PpcE) that cannot couple e-/H+ transfer in such range have three or four aromatic side chains. In the previously reported PpcAF15L mutant,14 carrying only one aromatic side chain, also the e-/H+ transfer coupling is affected. It is not clear in what way the aromatic rings present in the core of the protein influence the redox-Bohr effect and e-/H+ transfer coupling, but it is very striking that the cytochromes within this family having two internal aromatic side chains are capable of e-/H+ transfer, whereas the cytochromes carrying one internal aromatic side chain (as in PpcAF15L mutant), or three or four internal aromatic side chains (as in PpcAL6F mutant or PpcB or PpcE) are not. Only future work can establish the exact control exerted by the aromatic residues in these positions on the heme redox properties in control of this behavior.

CONCLUSIONS In the present work, we studied the role of residue 6 in the modulation of the redox-Bohr effect on the family of triheme cytochromes in Geobacter sulfurreducens. Cytochromes PpcA and PpcD showed important redox-Bohr effect compared to PpcB and PpcE within the physiological range for G. sulfurreducens growth. Comparison of the amino acid sequences of the cytochromes shows that the residue 6 is a leucine in both PpcA and PpcD, whereas the same residue is a phenylalanine in PpcB and PpcE. The residue 6 is located in the vicinity of the redox-Bohr center, which was assigned to propionate 13 of heme IV (P13IV). The residue 6 is a leucine in PpcA and PpcD, whereas it is a phenylalanine in PpcB and PpcE. This residue was replaced by phenylalanine in PpcA (PpcAL6F) and by leucine in PpcB and PpcE (PpcBF6L and PpcEF6L). The results show that the replacement of Leu6 by phenylalanine leads to a dramatic decrease in the redox-Bohr effect in PpcAL6F, whereas the reverse substitution yielded an increase in the redox-Bohr effect in both PpcB and PpcE. Moreover, the detailed thermodynamic characterization of PpcAL6F mutant showed that Leu6 is crucial to allow the native cytochrome displaying preferential e-/H+ transfer pathways. The structure of PpcAL6F mutant sheds light on the role of Leu6 residue in the regulation of the redox networks 15 ACS Paragon Plus Environment


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that are critical for the functional mechanism of the native cytochrome. The local structural changes in the vicinity of heme IV by replacing Leu6 by phenylalanine increased the heme solvent exposure considerably and had an effect in lowering its reduction potential. Moreover, the analysis of the effect of the pH on the chemical shift of mutant amide signals allowed mapping of the pH-linked conformational changes caused by protonation/deprotonation of the redox-Bohr center. The overall effect led to a decrease in the pKa value of the redox-Bohr center in the mutant, which affected the distribution of the dominant microstates along the protein redox cycle. The work presented allowed us to identify for the first time the structural determinants that explain how proteins from the same family with high structural homology can fine tune their properties by positioning suitable residues in key places to achieve different functional mechanisms. The functional and structural characterization of triheme cytochrome mutants from G. sulfurreducens can be explored in the future to develop strains containing cytochromes that favor e-/H+ transfer mechanisms within the physiological range.

ACKNOWLEDGMENTS Prof. D.L. Turner is acknowledged for providing us the software programs used to calculate the thermodynamic parameters and the solution structure. This work was supported by project grants: PTDC/BBB-BQB/3554/2014

(to

CAS),

SFRH/BD/89701/2012; SFRH/BD/86439/2012

and

SFRH/BD/61952/2009 (to JMD, APF and MAS, respectively), and UID/Multi/04378/2013 from Fundação para a Ciência e a Tecnologia, Portugal. The NMR spectrometers are part of The National NMR Facility, supported by Fundação para a Ciência e a Tecnologia (RECI/BBB-BQB/0230/2012). 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. DEAC02-06CH11357.


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Supporting Information Available: Supplementary table with chemical shifts of PpcAL6F mutant and wild-type cytochromes in the reduced state (Table S1). Supplementary figures with electronic distribution for the different microstates in triheme cytochromes (Figure S1), examples of 2D-1H EXSY NMR spectra obtained at intermediate oxidation stages (Figure S2) and sequential NOE connectivities together with average pairwise backbone and heavy atom rmsd values per residue of the family obtained for the solution structure (Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org.



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(2)

Lovley, D. R. Powering microbes with electricity: Direct electron transfer from electrodes to microbes. Environ. Microbiol. Rep. 2011, 3, 27-35.

(3)

Nealson, K. H.; Belz, A.; McKee, B. Breathing metals as a way of life: Geobiology in action. Antonie Van Leeuwenhoek. 2002, 81, 215-222.

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Lovley, D. R. Bioremediation. Anaerobes to the rescue. Science. 2001, 293, 1444-1446.

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Lovley, D. R. Bug juice: Harvesting electricity with microorganisms. Nat. Rev. Microbiol. 2006, 4, 497-508.

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Hau, H. H.; Gralnick, J. A. Ecology and biotechnology of the genus Shewanella. Annu. Rev. Microbiol. 2007, 61, 237-258.

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Mahadevan, R.; Bond, D. R.; Butler, J. E.; Esteve-Núñez, A.; Coppi, M. V.; Palsson, B. O.; Schilling, C. H.; Lovley, D. R. Characterization of metabolism in the Fe(III)-reducing organism Geobacter sulfurreducens by constraint-based modeling. Appl. Environ. Microbiol. 2006, 72, 1558-1568.

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Srinivasan, K.; Mahadevan, R. Characterization of proton production and consumption associated with microbial metabolism. BMC Biotechnol. 2010, 10, 2.

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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.


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(10)

Lloyd, J. R.; Leang, C.; Hodges Myerson, A. L.; Coppi, M. V.; Ciufo, S.; Methe, B.; Sandler, S. J.; Lovley, D. R. Biochemical and genetic characterization of PpcA, a periplasmic c-type cytochrome in Geobacter sulfurreducens. Biochem. J. 2003, 369, 153-161.

(11)

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.

(12)

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.

(13)

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.

(14)

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.

(15)

Morgado, L.; Dantas, J. M.; Simões, T.; Londer, Y. Y.; Pokkuluri, P. R.; Salgueiro, C. A. Role of Met(58) in the regulation of electron/proton transfer in trihaem cytochrome PpcA from Geobacter sulfurreducens. Biosci. Rep. 2013, 33, 11-22.

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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.



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Morgado, L.; Lourenço, S.; Londer, Y. Y.; Schiffer, M.; Pokkuluri, P. R.; Salgueiro, C. 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.

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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, 1-9.

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Pokkuluri, P. R.; Londer, Y. Y.; Yang, X.; Duke, N. E.; Erickson, J.; Orshonsky, V.; Johnson, G.; Schiffer, M. Structural characterization of a family of cytochromes c7 involved in Fe(III) respiration by Geobacter sulfurreducens. Biochim. Biophys. Acta. 2010, 1797, 222-232.

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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.

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Pokkuluri, P. R.; Yang, X.; Londer, Y. Y.; Schiffer, M. Pitfalls in the interpretation of structural changes in mutant proteins from crystal structures. J. Struct. Funct. Genomics. 2012, 13, 227-232.

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Pessanha, M.; Londer, Y. Y.; Long, W. C.; Erickson, J.; Pokkuluri, P. R.; Schiffer, M.; Salgueiro, C. A. Redox characterization of Geobacter sulfurreducens cytochrome c7: physiological relevance of the conserved residue F15 probed by site-specific mutagenesis. Biochemistry. 2004, 43, 9909-9917.

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Morgado, L.; Bruix, M.; Londer, Y. Y.; Pokkuluri, P. R.; Schiffer, M.; Salgueiro, C. A. Redox-linked conformational changes of a multiheme cytochrome from Geobacter sulfurreducens. Biochem. Biophys. Res. Commun. 2007, 360, 194-198.

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Morgado, L.; Paixao, 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.


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Fernandes, A. P.; Couto, I.; Morgado, L.; Londer, Y. Y.; Salgueiro, C. A. Isotopic labeling of c-type multiheme cytochromes overexpressed in E. coli. Prot. Expr. Purif. 2008, 59, 182-188.

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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.

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Koradi, R.; Billeter, M.; Wüthrich, K. MOLMOL: A program for display and analysis of macromolecular structures. J. Mol. Graph. 1996, 14, 51-55.

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Moss, G. P. Nomenclature of tetrapyrroles. Recommendations 1986 IUPAC-IUB joint commission on biochemical nomenclature (JCBN). Eur. J. Biochem. 1988, 178, 277-328.



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Table 1. Thermodynamic Parameters for PpcAL6F Mutant a Energy (meV) Heme I

Heme III

Heme IV

Redox-Bohr center

-168 (4)

22 (3)

7 (3)

-21 (6)

-154 (4)

33 (3)

-18 (6)

-140 (4)

-39 (5)

PpcAL6F Heme I Heme III Heme IV Redox-Bohr center

427 (10)

PpcA Heme I

-154 (5)

Heme III Heme IV

27 (2)

16 (3)

-32 (4)

-138 (5)

41 (3)

-31 (4)

-125 (5)

-58 (4)

Redox-Bohr center

495 (8)

a

For comparison, the values previously obtained for PpcA13 were also included. For each cytochrome, the fully reduced and protonated protein was taken as reference. Diagonal values (in bold) correspond to oxidation energies of the hemes and deprotonating energy of the redox-Bohr center. Off-diagonal values are the redox (heme-heme) and redox-Bohr (hemeproton) interaction energies. All energies are reported in meV, with standard errors given in parenthesis.


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Table 2. Macroscopic pKa Values of the Redox-Bohr Center for PpcAL6F in the Reduced and Oxidized States a pKa Oxidation stage PpcAL6F

PpcA

Reduced

7.4

8.6

Oxidized

6.1

6.5

pKa

1.3

2.1

a

For comparison, the values previously obtained for PpcA 13 were also included. The values were calculated from the parameters listed in Table 1. Following the nomenclature for the pairwise interacting centers model26, the pKa of the reduced proteins is given by gBF/(2.3RT) and the pKa of the 3   oxidized ones is given by  g B + ∑ giB  F/(2.3RT), i =1   where gB and giB are the deprotonating and interaction energies of the redox-Bohr center, respectively.



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Table 3. Summary of Restraint Violations and Quality Analysis of the Final Family of Solution Structures for PpcAL6F Parameter Type of distance restraint Intra-residue Sequential Medium range (2 ≤ |i - j| < 5) Long range (|i - j| ≥ 5) Total

769 539 504 586 2398 (1023 lov + 1375 upv)a

Upper distance limit violations Average maximum Number of consistent violations (> 0.2Å)

0.23 ± 0.07 0

Lower distance limit violations Average maximum Number of consistent violations (> 0.2Å)

0.25 ± 0.03 0

Van der Waals violations Average maximum Number of consistent violations (> 0.2Å)

0.23 ± 0.04 0

Ramachandran plot (%)b Most favoured regions Additionally allowed regions Generously allowed regions Disallowed regions Stereospecific assignments c Precision Average pairwise rmsd backbone (Å) Average pairwise rmsd heavy atoms (Å)

62.1 37.4 0.5 0.0 33

0.33 ± 0.07 0.96 ± 0.09

a

The total extent of assignment for was 90% (excluding carboxyl, amino and hydroxyl groups) b Values obtained with PROCHECK-NMR c Analysis with GLOMSA


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Table 4. Heme Geometry for PpcAL6F and PpcA12 Cytochromes in Solution a PpcA PpcAL6F Heme Fe-Fe distance (Å) I-III 11.7 I-IV 19.0 III-IV 12.6

11.6 18.5 13.4

Angle between heme planes (°) I-III 82 I-IV 27 III-IV 74

74 25 75

a

The values for both proteins were obtained for the lowest-energy NMR structure.



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Table 5. Number of Aromatic Side Chains, Excluding Heme Axial Ligands, for Cytochromes PpcA, PpcB, PpcD and PpcE from Geobacter sulfurreducens Cytochrome Residue Number of aromatic side chains 6 15 41 45 PpcA L F F M 2 PpcD L F M W 2 PpcB F F F M 3 PpcE F F F Y 4


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Figure 1. (A) Alignment of amino acid sequences PpcA-E. The conserved residues in the proteins are boxed: heme attached (gray) and non-heme attached residues (black). The specific heme and the respective attached residues are indicated on the bottom of the PpcE amino acid sequence. The percentage of the sequence identity for each cytochrome in relation to PpcA is also indicated. (B) Comparison of the heme core region containing residues 6 in PpcA (PDB ID: 2LDO12), PpcB (PDB ID: 3BXU19), PpcD (PDB ID: 3H4N19) and PpcE (PDB ID: 3H3419). The structure of PpcA is represented as a Cα ribbon colored gray. The hemes are numbered by the order of attachment to the CXXCH motif in the polypeptide chain and colored orange, light blue, yellow and dark blue in the structures of PpcA, PpcB, PpcD and PpcE, respectively. The side chains of the residue 6 (leucine in PpcA/PpcD and phenylanine in PpcB/PpcE) are also indicated with the same color code. (C) 27 ACS Paragon Plus Environment


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Expansion and superimposing of the heme core region containing the side chain of Leu6 in PpcA and PpcD. (D) Expansion and superimposing of the heme core region containing the side chain of Phe6 in PpcB and PpcE. In both expansions the redox-Bohr center (heme IV propionate 13, P13IV) is labeled. Structures

were

superimposed

in

MOLMOL27

using

backbone

atoms.

Figure 2. 2D 1H,15N-HSQC NMR spectra of PpcAL6F (blue contours) and PpcA (black contours). The most affected signals are connected by a straight line in the spectrum. The inset represents the weighted average of 1H and 15N chemical shifts (∆δavg) of each backbone amide.


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Figure 3. Fitting of the thermodynamic model to the experimental data for PpcAL6F. The black solid lines are the result of the simultaneous fitting of the NMR and visible data. The three left panels show the pH dependence of heme methyl chemical shifts at oxidation stages 1 (), 2 (), and 3 (). The chemical shift of the heme methyls in the fully reduced stage (stage 0) are not plotted since they are unaffected by the pH. The chemical shifts correspondent to the oxidation stage 0 for each heme methyl are 2.81, 4.15 and 3.51 ppm for 121CH3I, 71CH3III and 121CH3IV, respectively (see also Supplementary Table S1). Oxidation stages 1, 2 and 3 correspond to the first, second and third electron transfer respectively, as described in the Supplementary Figure S1. The solid red lines in each panel represent the best fit for the wild-type protein13. The right lower panel corresponds to the



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reduced fractions determined by visible spectroscopy at pH 7 () and pH 8 ().The heme spatial disposition in PpcAL6F and the IUPAC nomenclature for tetrapyrroles28 are illustrated.


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Figure 4. Order of oxidation of the hemes and functional mechanism of PpcAL6F and wild-type cytochromes. (A) Oxidized fractions of PpcAL6F heme groups (solid lines) and wild-type (dashed lines) at pH 7.5. The eapp values of the hemes for PpcAL6F and PpcA are indicated in the solid and dashed insets, respectively. (B) Molar fractions of each microstate for PpcAL6F (left panel) and 31 ACS Paragon Plus Environment


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wild-type (right panel) at pH 7.5. In both panels the curves were calculated as a function of the solution reduction potential using the parameters listed in Table 1. Solid and dashed lines indicate the protonated and deprotonated microstates, respectively. For clarity only the relevant microstates are labeled (see Supplementary Figure S1). The values for PpcA were previously obtained13 and are included in both panels for comparison.


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Figure 5. Redox titrations followed by visible spectroscopy for PpcBF6L and PpcEF6L at pH 7 () and 8 (). Blue solid lines indicate the results of the fits for the macroscopic reduction potentials, as described in Supplementary Figure S1. The midpoint reduction potentials (i.e., the point at which the oxidized and reduced fractions of the protein are equal) are indicated in each panel. The redox titrations for PpcA, PpcB, PpcD and PpcE were previously obtained.13 33 ACS Paragon Plus Environment


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Figure 6. Solution structure of PpcAL6F at pH 7.1. (A) Number of restraints per residue. Bars are white, light gray, dark gray and black for intra residue, sequential, medium and long range restraints, respectively. Residues 30, 54 and 68 also include restraints to hemes I, III and IV, respectively. (B) Overlay of the 20 lowest energy NMR solution structures. Superimposition was performed using all the heavy-atoms. The peptide chain and the hemes are colored blue and gray, respectively. (C) Ribbon diagram of PpcAL6F structure with a few residues labeled along the path of the polypeptide chain. The N- and C-termini are indicated in both figures, which were produced using MOLMOL27.


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Figure 7. Comparison of PpcAL6F and PpcA solution structures. Structures were superimposed in MOLMOL27 using backbone atoms (rmsd of 1.20 Å). (A) PpcAL6F versus PpcA solution structure families (PDB ID: 2LDO12). PpcAL6F and PpcA solution structures are colored blue and gray, respectively. (B) Average rmsd between the mean of each pair of structures.



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Figure 8. Comparison of the pH-linked conformational changes between PpcAL6F and PpcA. (A) Weighted average of 1H and

15

N chemical

shifts (δavg) between pH 5.5 and 9.5. The values obtained for PpcAL6F and PpcA are represented by open and close circles, respectively. Residues showing large pH-dependent shifts are mapped on PpcAL6F solution structure. (B) and (C) pH titration data of the most affected PpcAL6F NH signals (solid lines). The dashed lines in each panel represent the best fit for the wild-type protein12. In the expansion of each 1

H,15N-HSQC NMR spectrum the pH increases from yellow to green.

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Table of Contents (TOC) Image


Unveiling the Structural Basis That Regulates the Energy Transduction

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