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

PpcA is the most abundant member of a family of five triheme cytochromes c 7 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.

[1]  L. Morgado,et al.  Role of Met58 in the regulation of electron/proton transfer in trihaem cytochrome PpcA from Geobacter sulfurreducens , 2012, Bioscience reports.

[2]  M. Bruix,et al.  Fine Tuning of Redox Networks on Multiheme Cytochromes from Geobacter sulfurreducens Drives Physiological Electron/Proton Energy Transduction , 2012, Bioinorganic chemistry and applications.

[3]  G. Reguera,et al.  Electron Donors Supporting Growth and Electroactivity of Geobacter sulfurreducens Anode Biofilms , 2011, Applied and Environmental Microbiology.

[4]  M. Bruix,et al.  Backbone, side chain and heme resonance assignments of the triheme cytochrome PpcD from Geobacter sulfurreducens , 2011, Biomolecular NMR Assignments.

[5]  M. Bruix,et al.  Thermodynamic characterization of a triheme cytochrome family from Geobacter sulfurreducens reveals mechanistic and functional diversity. , 2010, Biophysical journal.

[6]  N. Duke,et al.  Structural characterization of a family of cytochromes c(7) involved in Fe(III) respiration by Geobacter sulfurreducens. , 2010, Biochimica et biophysica acta.

[7]  D. Lovley,et al.  Evolution of electron transfer out of the cell: comparative genomics of six Geobacter genomes , 2010, BMC Genomics.

[8]  Kenneth H. Williams,et al.  Proteogenomic Monitoring of Geobacter Physiology during Stimulated Uranium Bioremediation , 2009, Applied and Environmental Microbiology.

[9]  Alla Lapidus,et al.  The genome sequence of Geobacter metallireducens: features of metabolism, physiology and regulation common and dissimilar to Geobacter sulfurreducens , 2009, BMC Microbiology.

[10]  Richard D. Smith,et al.  Proteome of Geobacter sulfurreducens grown with Fe(III) oxide or Fe(III) citrate as the electron acceptor. , 2008, Biochimica et biophysica acta.

[11]  N. Duke,et al.  Structural insights into the modulation of the redox properties of two Geobacter sulfurreducens homologous triheme cytochromes. , 2008, Biochimica et biophysica acta.

[12]  M. Schiffer,et al.  Redox-linked conformational changes of a multiheme cytochrome from Geobacter sulfurreducens. , 2007, Biochemical and biophysical research communications.

[13]  Laurie N. DiDonato,et al.  Importance of c-Type cytochromes for U(VI) reduction by Geobacter sulfurreducens , 2007, BMC Microbiology.

[14]  M. Schiffer,et al.  Thermodynamic characterization of triheme cytochrome PpcA from Geobacter sulfurreducens: evidence for a role played in e-/H+ energy transduction. , 2006, Biochemistry.

[15]  Richard D. Smith,et al.  The proteome of dissimilatory metal-reducing microorganism Geobacter sulfurreducens under various growth conditions. , 2006, Biochimica et biophysica acta.

[16]  R. Louro,et al.  Distance dependence of interactions between charged centres in proteins with common structural features , 2004, FEBS letters.

[17]  M. Schiffer,et al.  Redox characterization of Geobacter sulfurreducens cytochrome c7: physiological relevance of the conserved residue F15 probed by site-specific mutagenesis. , 2004, Biochemistry.

[18]  N. Duke,et al.  Family of cytochrome c7-type proteins from Geobacter sulfurreducens: structure of one cytochrome c7 at 1.45 A resolution. , 2004, Biochemistry.

[19]  J A Eisen,et al.  Genome of Geobacter sulfurreducens: Metal Reduction in Subsurface Environments , 2003, Science.

[20]  M. Schiffer,et al.  Production and preliminary characterization of a recombinant triheme cytochrome c(7) from Geobacter sulfurreducens in Escherichia coli. , 2002, Biochimica et biophysica acta.

[21]  T. Catarino,et al.  NMR studies of cooperativity in the tetrahaem cytochrome c3 from Desulfovibrio vulgaris. , 1996, European journal of biochemistry.

[22]  H. Santos,et al.  Assignment of the redox potentials to the four haems in Desulfovibrio vulgaris cytochrome c 3 by 2D‐NMR , 1992, FEBS letters.

[23]  G. P. Moss.  Nomenclature of tetrapyrroles , 1988 .

[24]  H. Santos,et al.  NMR studies of electron transfer mechanisms in a protein with interacting redox centres: Desulfovibrio gigas cytochrome c3. , 1984, European journal of biochemistry.

[25]  J. E. Merritt,et al.  IUPAC-IUB joint commission on biochemical nomenclature (JCBN). Nomenclature of tetrapyrroles. Recommendations 1978. , 1980, European journal of biochemistry.

[26]  M. Schiffer,et al.  Revealing the structural origin of the redox-Bohr effect: the first solution structure of a cytochrome from Geobacter sulfurreducens. , 2012, The Biochemical journal.

[27]  M. Schiffer,et al.  Pivotal role of the strictly conserved aromatic residue F15 in the cytochrome c7 family , 2011, JBIC Journal of Biological Inorganic Chemistry.

[28]  M. Bruix,et al.  Backbone, side chain and heme resonance assignments of the triheme cytochrome PpcA from Geobacter sulfurreducens , 2011, Biomolecular NMR assignments.

[29]  Tian Zhang,et al.  Geobacter: the microbe electric's physiology, ecology, and practical applications. , 2011, Advances in microbial physiology.

[30]  C. Leang,et al.  Biochemical and genetic characterization of PpcA, a periplasmic c-type cytochrome in Geobacter sulfurreducens. , 2003, The Biochemical journal.

[31]  H. Schulz,et al.  Overproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichia coli. , 1998, Biochemical and biophysical research communications.

[32]  G. P. Moss,et al.  Nomenclature of tetrapyrroles. Recommendations 1986 IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN). , 1988, European journal of biochemistry.