Effect of Hydrogen-Bond Networks in Controlling Reduction Potentials in Desulfovibrio vulgaris (Hildenborough) Cytochrome c3 Probed by Site-Specific Mutagenesis

• Published in: Biochemistry (Easton) Vol. 40; no. 32; pp. 9709 - 9716
• Main Authors: SALGUEIRO, Carlos A., DA COSTA, Patrícia N., TURNER, David L., MESSIAS, Ana C., VAN DONGEN, Walter M. A. M., SARAIVA, Lígia M., XAVIER, António V.
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 14.08.2001
• Subjects: Biochemie; Biochemistry; Cytochrome c Group - chemistry; Cytochrome c Group - genetics; Desulfovibrio vulgaris - metabolism; Hydrogen Bonding; Hydrogen-Ion Concentration; Models, Molecular; Mutagenesis, Site-Directed; Nuclear Magnetic Resonance, Biomolecular; Oxidation-Reduction; VLAG
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi010330b

Cytochromes C3 isolated from Desulfovibrio spp. are periplasmic proteins that play a central role in energy transduction by coupling the transfer of electrons and protons from hydrogenase. Comparison between the oxidized and reduced structures of cytochrome C3 isolated from Desulfovibrio vulgaris (Hildenborough) show that the residue threonine 24, located in the vicinity of heme III, reorients between these two states [Messias, A. C., Kastrau, D. H. W., Costa, H. S., LeGall, J., Turner, D. L., Santos, H., and Xavier, A. V. (1998) J. Mol. Biol. 281, 719-739]. Threonine 24 was replaced with valine by site-directed mutagenesis to elucidate its effect on the redox properties of the protein. The NMR spectra of the mutated protein are very similar to those of the wild type, showing that the general folding and heme core architecture are not affected by the mutation. However, thermodynamic analysis of the mutated cytochrome reveals a large alteration in the microscopic reduction potential of heme III (75 and 106 mV for the protonated forms of the fully reduced and oxidized states, respectively). The redox interactions involving this heme are also modified, while the remaining heme-heme interactions and the redox-Bohr interactions are less strongly affected. Hence, the order of oxidation of the hemes in the mutated cytochrome is different from that in the wild type, and it has a higher overall affinity for electrons. This is consistent with the replacement of threonine 24 by valine preventing the formation of a network of hydrogen bonds, which stabilizes the oxidized state. The mutated protein is unable to perform a concerted two-electron step between the intermediate oxidation stages, 1 and 3, which can occur in the wild-type protein. Thus, replacing a single residue unbalances the global network of cooperativities tuned to control thermodynamically the directionality of the stepwise electron transfer and may affect the functionality of the protein.