Dantas, JM, Portela PC, Fernandes AP, Londer YY, Yang X, Duke NEC, Schiffer M, Pokkuluri RP, Salgueiro CA.
2019.
Structural and Functional Relevance of the Conserved Residue V13 in the Triheme Cytochrome PpcA from Geobacter sulfurreducens. The Journal of Physical Chemistry B. 123:3050-3060., Number 14
AbstractThe 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 well-established 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 this 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 were 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 the 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, were 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 more distinctive conformations, 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 ensure a unique conformation of the protein and the order of oxidation of the hemes, endowing the cytochrome PpcA with the optimal functional properties necessary to ensure effectiveness in the extracellular electron transfer respiratory pathways of G. sulfurreducens.
Dantas, JM, Morgado L, Pokkuluri PR, Turner DL, Salgueiro CA.
2013.
Solution structure of a mutant of the triheme cytochrome PpcA from Geobacter sulfurreducens sheds light on the role of the conserved aromatic residue F15. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1827(4):484-492.
AbstractExtracellular electron transfer is one of the physiological hallmarks of Geobacteraceae. Most of the Geobacter species encode for more than 100 c-type cytochromes which are, in general, poorly conserved between individual species. An exception to this is the PpcA family of periplasmic triheme c-type cytochromes, which are the most abundant proteins in these bacteria. The functional characterization of PpcA showed that it has the necessary properties to couple electron/proton transfer, a fundamental step for ATP synthesis. The detailed thermodynamic characterization of a PpcA mutant, in which the strictly conserved residue phenylalanine 15 was replaced by leucine, showed that the global redox network of cooperativities among heme groups is altered, preventing the mutant from performing a concerted electron/proton transfer. In this work, we determined the solution structure of PpcA F15L mutant in the fully reduced state using NMR spectroscopy by producing 15N-labeled protein. In addition, pH-dependent conformational changes were mapped onto the structure. The mutant structure obtained is well defined, with an average pairwise root-mean-square deviation of 0.36 Å for the backbone atoms and 1.14 Å for all heavy atoms. Comparison between the mutant and wild-type structures elucidated the contribution of phenylalanine 15 in the modulation of the functional properties of PpcA.
Dantas, JM, Salgueiro CA, Bruix M.
2015.
Backbone, side chain and heme resonance assignments of the triheme cytochrome PpcD from Geobacter sulfurreducens. Biomol NMR Assign. 9(1):211-214.
AbstractGene knock-out studies on Geobacter sulfurreducens (Gs) cells showed that the periplasmic triheme cytochrome PpcD is involved in respiratory pathways leading to the extracellular reduction of Fe(III) and U(VI) oxides. More recently, it was also shown that the gene encoding for PpcD has higher transcript abundance when Gs cells utilize graphite electrodes as sole electron donors to reduce fumarate. This sets PpcD as the first multiheme cytochrome to be involved in Gs respiratory pathways that bridge the electron transfer between the cytoplasm and cell exterior in both directions. Nowadays, extracellular electron transfer (EET) processes are explored for several biotechnological applications, which include bioremediation, bioenergy and biofuel production. Therefore, the structural characterization of PpcD is a fundamental step to understand the mechanisms underlying EET. However, compared to non-heme proteins, the presence of numerous proton-containing groups in the redox centers presents additional challenges for protein signal assignment and structure calculation. Here, we report the complete assignment of the heme proton signals together with 1H, 13C and 15N backbone and side chain assignments of the reduced form of PpcD.
Dantas, JM, Kokhan O, Pokkuluri RP, Salgueiro CA.
2015.
Molecular interaction studies revealed the bifunctional behavior of triheme cytochrome PpcA from Geobacter sulfurreducens toward the redox active analog of humic substances. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1847:1129-1138., Number 10
AbstractAbstract Humic substances (HS) constitute a significant fraction of natural organic matter in terrestrial and aquatic environments and can act as terminal electron acceptors in anaerobic microbial respiration. Geobacter sulfurreducens has a remarkable respiratory versatility and can utilize the \{HS\} analog anthraquinone-2,6-disulfonate (AQDS) as a terminal electron acceptor or its reduced form (AH2QDS) as an electron donor. Previous studies set the triheme cytochrome PpcA as a key component for \{HS\} respiration in G. sulfurreducens, but the process is far from fully understood. In this work, \{NMR\} chemical shift perturbation measurements were used to map the interaction region between PpcA and AH2QDS, and to measure their binding affinity. The results showed that the \{AH2QDS\} binds reversibly to the more solvent exposed edge of PpcA heme IV. The \{NMR\} and visible spectroscopies coupled to redox measurements were used to determine the thermodynamic parameters of the PpcA:quinol complex. The higher reduction potential of heme İV\} (− 127 mV) compared to that of \{AH2QDS\} (− 184 mV) explains why the electron transfer is more favorable in the case of reduction of the cytochrome by the quinol. The clear evidence obtained for the formation of an electron transfer complex between \{AH2QDS\} and PpcA, combined with the fact that the protein also formed a redox complex with AQDS, revealed for the first time the bifunctional behavior of PpcA toward an analog of the HS. Such behavior might confer selective advantage to G. sulfurreducens, which can utilize the \{HS\} in any redox state available in the environment for its metabolic needs.