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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.

ABSTRACT: 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.

SUBMITTER: Morgado L

PROVIDER: S-EPMC4143306 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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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.

Morgado Leonor L Lourenço Sílvia S Londer Yuri Y YY Schiffer Marianne M Pokkuluri P Raj PR Salgueiro Carlos A CA

PloS one 20140825 8

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 funct ...[more]

PMID: 25153891

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Project description:Multiheme cytochromes have been implicated in Geobacter sulfurreducens extracellular electron transfer (EET). These proteins are potential targets to improve EET and enhance bioremediation and electrical current production by G. sulfurreducens. However, the functional characterization of multiheme cytochromes is particularly complex due to the co-existence of several microstates in solution, connecting the fully reduced and fully oxidized states. Over the last decade, new strategies have been developed to characterize multiheme redox proteins functionally and structurally. These strategies were used to reveal the functional mechanism of G. sulfurreducens multiheme cytochromes and also to identify key residues in these proteins for EET. In previous studies, we set the foundations for enhancement of the EET abilities of G. sulfurreducens by characterizing a family of five triheme cytochromes (PpcA-E). These periplasmic cytochromes are implicated in electron transfer between the oxidative reactions of metabolism in the cytoplasm and the reduction of extracellular terminal electron acceptors at the cell's outer surface. The results obtained suggested that PpcA can couple e(-)/H(+) transfer, a property that might contribute to the proton electrochemical gradient across the cytoplasmic membrane for metabolic energy production. The structural and functional properties of PpcA were characterized in detail and used for rational design of a family of 23 single site PpcA mutants. In this review, we summarize the functional characterization of the native and mutant proteins. Mutants that retain the mechanistic features of PpcA and adopt preferential e(-)/H(+) transfer pathways at lower reduction potential values compared to the wild-type protein were selected for in vivo studies as the best candidates to increase the electron transfer rate of G. sulfurreducens. For the first time G. sulfurreducens strains have been manipulated by the introduction of mutant forms of essential proteins with the aim to develop and improve bioelectrochemical technologies.

Project description:The monoheme outer membrane cytochrome F (OmcF) from Geobacter sulfurreducens plays an important role in Fe(III) reduction and electric current production. The electrochemical characterization of this cytochrome has shown that its redox potential is modulated by the solution pH (redox-Bohr effect) endowing the protein with the necessary properties to couple electron and proton transfer in the physiological range. The analysis of the OmcF structures in the reduced and oxidized states showed that with the exception of the side chain of histidine 47 (His47), all other residues with protonatable side chains are distant from the heme iron and, therefore, are unlikely to affect the redox potential of the protein. The protonatable site at the imidazole ring of His47 is in the close proximity to the heme and, therefore, this residue was suggested as the redox-Bohr center. In the present work, we tested this hypothesis by replacing the His47 with non-protonatable residues (isoleucine - OmcFH47I and phenylalanine - OmcFH47F). The structure of the mutant OmcFH47I was determined by X-ray crystallography to 1.13 Å resolution and showed only minimal changes at the site of the mutation. Both mutants were 15N-labeled and their overall folding was confirmed to be the same as the wild-type by NMR spectroscopy. The pH dependence of the redox potential of the mutants was measured by cyclic voltammetry. Compared to the wild-type protein, the magnitude of the redox-Bohr effect in the mutants was smaller, but not fully abolished, confirming the role of His47 on the pH modulation of OmcF's redox potential. However, the pH effect on the heme substituents' NMR chemical shifts suggested that the heme propionate P13 also contributes to the overall redox-Bohr effect in OmcF. In physiological terms, the contribution of two independent acid-base centers to the observed redox-Bohr effect confers OmcF a higher versatility to environmental changes by coupling electron/proton transfer within a wider pH range.

Project description:Our previous study showed that CYP105D7, a substrate-promiscuous P450, catalyzes the hydroxylation of 1-deoxypentalenic acid, diclofenac, naringenin, and compactin. In this study, 14 steroid compounds were screened using recombinant Escherichia coli cells harboring genes encoding CYP105D7 and redox partners (Pdx/Pdr, RhFRED, and FdxH/FprD), and the screening identified steroid A-ring 2?- and D-ring 16?-hydroxylation activity. Wild-type CYP105D7 was able to catalyze the hydroxylation of five steroids (testosterone, progesterone, 4-androstene-3,17-dione, adrenosterone, and cortisone) with low (<10%) conversion rates. Structure-guided site-directed mutagenesis of arginine residues around the substrate entrance and active site showed that the R70A and R190A single mutants and an R70A/R190A double mutant exhibited greatly enhanced conversion rates for steroid hydroxylation. For the conversion of testosterone in particular, the R70A/R190A mutant's k cat/Km values increased 1.35-fold and the in vivo conversion rates increased significantly by almost 9-fold with high regio- and stereoselectivity. Molecular docking analysis revealed that when Arg70 and Arg190 were replaced with alanine, the volume of the substrate access and binding pocket increased 1.08-fold, which might facilitate improvement of the hydroxylation efficiency of steroids.IMPORTANCE Cytochrome P450 monooxygenases (P450s) are able to introduce oxygen atoms into nonreactive hydrocarbon compounds under mild conditions, thereby offering significant advantages compared to chemical catalysts. Promiscuous P450s with broad substrate specificity and reaction diversity have significant potential for applications in various fields, including synthetic biology. The study of the function, molecular mechanisms, and rational engineering of substrate-promiscuous P450s from microbial sources is important to fulfill this potential. Here, we present a microbial substrate-promiscuous P450, CYP105D7, which can catalyze hydroxylation of steroids. The loss of the bulky side chains of Arg70 and Arg190 in the active site and substrate entrance resulted in an up to 9-fold increase in the substrate conversion rate. These findings will support future rational and semirational engineering of P450s for applications as biocatalysts.

Dataset Information

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.

Publications

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.

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