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1.  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(8):e105566.
PpcA is the most abundant member of a family of five triheme cytochromes c7 in the bacterium Geobacter sulfurreducens (Gs) and is the most likely carrier of electrons destined for outer surface during respiration on solid metal oxides, a process that requires extracellular electron transfer. This cytochrome has the highest content of lysine residues (24%) among the family, and it was suggested to be involved in e−/H+ energy transduction processes. In the present work, we investigated the functional role of lysine residues strategically located in the vicinity of each heme group. Each lysine was replaced by glutamine or glutamic acid to evaluate the effects of a neutral or negatively charged residue in each position. The results showed that replacing Lys9 (located near heme IV), Lys18 (near heme I) or Lys22 (between hemes I and III) has essentially no effect on the redox properties of the heme groups and are probably involved in redox partner recognition. On the other hand, Lys43 (near heme IV), Lys52 (between hemes III and IV) and Lys60 (near heme III) are crucial in the regulation of the functional mechanism of PpcA, namely in the selection of microstates that allow the protein to establish preferential e−/H+ transfer pathways. The results showed that the preferred e−/H+ transfer pathways are only established when heme III is the last heme to oxidize, a feature reinforced by a higher difference between its reduction potential and that of its predecessor in the order of oxidation. We also showed that K43 and K52 mutants keep the mechanistic features of PpcA by establishing preferential e−/H+ transfer pathways at lower reduction potential values than the wild-type protein, a property that can enable rational design of Gs strains with optimized extracellular electron transfer capabilities.
doi:10.1371/journal.pone.0105566
PMCID: PMC4143306  PMID: 25153891
2.  Nonredundant Roles for Cytochrome c2 and Two High-Potential Iron-Sulfur Proteins in the Photoferrotroph Rhodopseudomonas palustris TIE-1 
Journal of Bacteriology  2014;196(4):850-858.
The purple bacterium Rhodopseudomonas palustris TIE-1 expresses multiple small high-potential redox proteins during photoautotrophic growth, including two high-potential iron-sulfur proteins (HiPIPs) (PioC and Rpal_4085) and a cytochrome c2. We evaluated the role of these proteins in TIE-1 through genetic, physiological, and biochemical analyses. Deleting the gene encoding cytochrome c2 resulted in a loss of photosynthetic ability by TIE-1, indicating that this protein cannot be replaced by either HiPIP in cyclic electron flow. PioC was previously implicated in photoferrotrophy, an unusual form of photosynthesis in which reducing power is provided through ferrous iron oxidation. Using cyclic voltammetry (CV), electron paramagnetic resonance (EPR) spectroscopy, and flash-induced spectrometry, we show that PioC has a midpoint potential of 450 mV, contains all the typical features of a HiPIP, and can reduce the reaction centers of membrane suspensions in a light-dependent manner at a much lower rate than cytochrome c2. These data support the hypothesis that PioC linearly transfers electrons from iron, while cytochrome c2 is required for cyclic electron flow. Rpal_4085, despite having spectroscopic characteristics and a reduction potential similar to those of PioC, is unable to reduce the reaction center. Rpal_4085 is upregulated by the divalent metals Fe(II), Ni(II), and Co(II), suggesting that it might play a role in sensing or oxidizing metals in the periplasm. Taken together, our results suggest that these three small electron transfer proteins perform different functions in the cell.
doi:10.1128/JB.00843-13
PMCID: PMC3911180  PMID: 24317397
3.  Correction: Protein Folding Modulates the Swapped Dimerization Mechanism of Methyl-Accepting Chemotaxis Heme Sensors 
PLoS ONE  2013;8(3):10.1371/annotation/0b01096f-01ab-4063-a15f-357dff5c0270.
doi:10.1371/annotation/0b01096f-01ab-4063-a15f-357dff5c0270
PMCID: PMC3586584
4.  Role of Met58 in the regulation of electron/proton transfer in trihaem cytochrome PpcA from Geobacter sulfurreducens 
Bioscience Reports  2012;33(1):e00002.
The bacterium Gs (Geobacter sulfurreducens) is capable of oxidizing a large variety of compounds relaying electrons out of the cytoplasm and across the membranes in a process designated as extracellular electron transfer. The trihaem cytochrome PpcA is highly abundant in Gs and is most probably the reservoir of electrons destined for the outer surface. In addition to its role in electron transfer pathways, we have previously shown that this protein could perform e−/H+ energy transduction. This mechanism is achieved by selecting the specific redox states that the protein can access during the redox cycle and might be related to the formation of proton electrochemical potential gradient across the periplasmic membrane. The regulatory role of haem III in the functional mechanism of PpcA was probed by replacing Met58, a residue that controls the solvent accessibility of haem III, with serine, aspartic acid, asparagine or lysine. The data obtained from the mutants showed that the preferred e−/H+ transfer pathway observed for PpcA is strongly dependent on the reduction potential of haem III. It is striking to note that one residue can fine tune the redox states that can be accessed by the trihaem cytochrome enough to alter the functional pathways.
doi:10.1042/BSR20120086
PMCID: PMC3522473  PMID: 23030844
electron transfer; Geobacter; multihaem cytochrome; NMR site-directed mutagenesis; EXSY, exchange spectroscopy; Gs, Geobacter sulfurreducens; HSQC, heteronuclear single-quantum coherence; NOESY, nuclear Overhauser enhancement spectroscopy; rmsd, root mean square deviation; 2D, two-dimensional
5.  Protein Folding Modulates the Swapped Dimerization Mechanism of Methyl-Accepting Chemotaxis Heme Sensors 
PLoS ONE  2012;7(9):e46328.
The periplasmic sensor domains GSU0582 and GSU0935 are part of methyl accepting chemotaxis proteins in the bacterium Geobacter sulfurreducens. Both contain one c-type heme group and their crystal structures revealed that these domains form swapped dimers with a PAS fold formed from the two protein chains. The swapped dimerization of these sensors is related to the mechanism of signal transduction and the formation of the swapped dimer involves significant folding changes and conformational rearrangements within each monomeric component. However, the structural changes occurring during this process are poorly understood and lack a mechanistic framework. To address this issue, we have studied the folding and stability properties of two distinct heme-sensor PAS domains, using biophysical spectroscopies. We observed substantial differences in the thermodynamic stability (ΔG = 14.6 kJ.mol−1 for GSU0935 and ΔG = 26.3 kJ.mol−1 for GSU0582), and demonstrated that the heme moiety undergoes conformational changes that match those occurring at the global protein structure. This indicates that sensing by the heme cofactor induces conformational changes that rapidly propagate to the protein structure, an effect which is directly linked to the signal transduction mechanism. Interestingly, the two analyzed proteins have distinct levels of intrinsic disorder (25% for GSU0935 and 13% for GSU0582), which correlate with conformational stability differences. This provides evidence that the sensing threshold and intensity of the propagated allosteric effect is linked to the stability of the PAS-fold, as this property modulates domain swapping and dimerization. Analysis of the PAS-domain shows that disorder segments are found either at the hinge region that controls helix motions or in connecting segments of the β-sheet interface. The latter is known to be widely involved in both intra- and intermolecular interactions, supporting the view that it's folding and stability are at the basis of the specificity and regulation of many types of PAS-containing signaling proteins.
doi:10.1371/journal.pone.0046328
PMCID: PMC3460858  PMID: 23029480
6.  Fine Tuning of Redox Networks on Multiheme Cytochromes from Geobacter sulfurreducens Drives Physiological Electron/Proton Energy Transduction 
The bacterium Geobacter sulfurreducens (Gs) can grow in the presence of extracellular terminal acceptors, a property that is currently explored to harvest electricity from aquatic sediments and waste organic matter into microbial fuel cells. A family composed of five triheme cytochromes (PpcA-E) was identified in Gs. These cytochromes play a crucial role by bridging the electron transfer from oxidation of cytoplasmic donors to the cell exterior and assisting the reduction of extracellular terminal acceptors. The detailed thermodynamic characterization of such proteins showed that PpcA and PpcD have an important redox-Bohr effect that might implicate these proteins in the e−/H+ coupling mechanisms to sustain cellular growth. The physiological relevance of the redox-Bohr effect in these proteins was studied by determining the fractional contribution of each individual redox-microstate at different pH values. For both proteins, oxidation progresses from a particular protonated microstate to a particular deprotonated one, over specific pH ranges. The preferred e−/H+ transfer pathway established by the selected microstates indicates that both proteins are functionally designed to couple e−/H+ transfer at the physiological pH range for cellular growth.
doi:10.1155/2012/298739
PMCID: PMC3415244  PMID: 22899897
7.  Purification and Characterization of OmcZ, an Outer-Surface, Octaheme c-Type Cytochrome Essential for Optimal Current Production by Geobacter sulfurreducens▿ †  
Applied and Environmental Microbiology  2010;76(12):3999-4007.
Previous studies have demonstrated that Geobacter sulfurreducens requires the c-type cytochrome OmcZ, which is present in large (OmcZL; 50-kDa) and small (OmcZS; 30-kDa) forms, for optimal current production in microbial fuel cells. This protein was further characterized to aid in understanding its role in current production. Subcellular-localization studies suggested that OmcZS was the predominant extracellular form of OmcZ. N- and C-terminal amino acid sequence analysis of purified OmcZS and molecular weight measurements indicated that OmcZS is a cleaved product of OmcZL retaining all 8 hemes, including 1 heme with the unusual c-type heme-binding motif CX14CH. The purified OmcZS was remarkably thermally stable (thermal-denaturing temperature, 94.2°C). Redox titration analysis revealed that the midpoint reduction potential of OmcZS is approximately −220 mV (versus the standard hydrogen electrode [SHE]) with nonequivalent heme groups that cover a large reduction potential range (−420 to −60 mV). OmcZS transferred electrons in vitro to a diversity of potential extracellular electron acceptors, such as Fe(III) citrate, U(VI), Cr(VI), Au(III), Mn(IV) oxide, and the humic substance analogue anthraquinone-2,6-disulfonate, but not Fe(III) oxide. The biochemical properties and extracellular localization of OmcZ suggest that it is well suited for promoting electron transfer in current-producing biofilms of G. sulfurreducens.
doi:10.1128/AEM.00027-10
PMCID: PMC2893489  PMID: 20400562

Results 1-7 (7)