No abstract included.

The NMR structure of the oxidised wild-type cytochrome c3 from Desulfovibrio vulgaris Hildenborough was determined in solution. Using a newly developed methodology, NMR data from the K45Q mutant was then grafted onto data from the wild-type protein to determine the structure in the region of the mutation. The structural origins of the redox-Bohr effect and haem–haem cooperativities are discussed with respect to the redox-related conformational changes observed in solution.

The facultative aerobic bacterium Geobacter sulfurreducens produces a small periplasmic c-type triheme cytochrome with 71 residues (PpcA) under anaerobic growth conditions, which is involved in the iron respiration. The thermodynamic properties of the PpcA redox centers and of a protonatable center were determined using NMR and visible spectroscopy techniques. The redox centers have negative and different reduction potentials (−162, −143, and −133 mV for heme I, III, and IV, respectively, for the fully reduced and protonated protein), which are modulated by redox interactions among the hemes (covering a range from 10 to 36 mV) and by redox−Bohr interactions (up to −62 mV) between the hemes and a protonatable center located in the proximity of heme IV. All the interactions between the four centers are dominated by electrostatic effects. The microscopic reduction potential of heme III is the one most affected by the oxidation of the other hemes, whereas heme IV is the most affected by the protonation state of the molecule. The thermodynamic properties of PpcA showed that pH strongly modulates the redox behavior of the individual heme groups. A preferred electron transfer pathway at physiologic pH is defined, showing that PpcA has the necessary thermodynamic properties to perform e-/H+ energy transduction, contributing to a H+ electrochemical potential gradient across the periplasmic membrane that drives ATP synthesis. PpcA is 46% identical in sequence to and shares a high degree of structural similarity with a periplasmic triheme cytochrome c7 isolated from Desulfuromonas acetoxidans, a bacterium closely related to the Geobacteracea family. However, the results obtained for PpcA are quite different from those published for D. acetoxidans c7, and the physiological consequences of these differences are discussed.

NMR and visible spectroscopy coupled to redox measurements were used to determine the equilibrium thermodynamic properties of the four haems in cytochrome c3 under conditions in which the protein was bound to ligands, the small anion phosphate and the protein rubredoxin with the iron in the active site replaced by zinc. Comparison of these results with data for the isolated cytochrome shows that binding of ligands causes only small changes in the reduction potentials of the haems and their pairwise interactions, and also that the redox-sensitive acid–base centre responsible for the redox–Bohr effect is essentially unaffected. Although neither of the ligands tested is a physiological partner of cytochrome c3, the small changes observed for the thermodynamic properties of cytochrome c3 bound to these ligands vs. the unbound state, indicate that the thermodynamic properties measured for the isolated protein are relevant for a physiological interpretation of the role of this cytochrome in the bioenergetic metabolism of Desulfovibrio.

NMR spectroscopy has been applied with great success to study electron transfer proteins
with multiple redox centers. This study aimed to elucidate the redox behavior the enzyme fumarate
reductase from Shewanella frigidimarina and particularly to reveal the electron transfer mechanism
from the N-terminal domain to the active center. We developed a new strategy encompassing the
acquisition of 1H-EXSY bidimensional spectra for observation of chemical exchange connectivities in
partially oxidized samples of fcc3, estimation of the paramagnetic chemical shifts expected for the
heme substituents and their comparison with NMR spectra obtained in the fully oxidized protein. This
study allowed obtaining the order of oxidation of the different groups (II-I-III, IV) and gave insights of
the functional mechanisms that allow fcc3 to efficiently transfer electrons from the N-terminal domain
to the active center.

Flavocytochrome c3 from Shewanella frigidimarina (fcc3) is a tetrahaem periplasmic protein of 64 kDa with fumarate reductase activity. This work reports the first example of NMR techniques applied to the assignment of the thermodynamic order of oxidation of the four individual haems for such large protein, expanding its applicability to a wide range of proteins. NMR data from partially and fully oxidised samples of fcc3 and a mutated protein with an axial ligand of haem IV replaced by alanine were compared with calculated chemical shifts, allowing the structural assignment of the signals and the unequivocal determination of the order of oxidation of the haems. As oxidation progresses the fcc3 haem domain is polarised, with haems I and II much more oxidised than haems III and IV, haem IV being the most reduced. Thus, during catalysis as an electron is taken by the flavin adenosine dinucleotide from haem IV, haem III is eager to re-reduce haem IV, allowing the transfer of two electrons to the active site.

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.

The structural basis for the pH dependence of the redox potential in the tetrahemic Desulfovibrio vulgaris (Hildenborough) cytochrome c3 was investigated by site-directed mutagenesis of charged residues in the vicinity of heme I. Mutation of lysine 45, located in the neighborhood of the propionates of heme I, by uncharged residues, namely threonine, glutamine and leucine, was performed. The replacement of a conserved charged residue, aspartate 7, present in the N-terminal region and near heme I was also attempted. The analysis of the redox interactions as well as the redox-Bohr behavior of the mutated cytochromes c3 allowed the conclusion that residue 45 has a functional role in the control of the pKa of the propionate groups of heme I and confirms the involvement of this residue in the redox-Bohr effect.