The tetraheme cytochrome c3 is a small metalloprotein with ca. 13,000 Da found in sulfate-reducing bacteria, which is believed to act as a partner of hydrogenase. The three-dimensional structure of the oxidized and reduced forms of cytochrome c3 from Desulfovibrio desulfuricans ATCC 27774 at pH 7.6 were determined using high-resolution X-ray crystallography and were compared with the previously determined oxidized form at pH 4.0. Theoretical calculations were performed with both structures, using continuum electrostatic calculations and Monte Carlo sampling of protonation and redox states, in order to understand the molecular basis of the redox-Bohr and cooperativity effects related to the coupled transfer of electrons and protons. We were able to identify groups that showed redox-linked conformational changes. In particular, Glu61, His76, and propionate D of heme II showed important contributions to the redox-cooperativity, whereas His76, propionate A of heme I, and propionate D of heme IV were the key residues for the redox-Bohr effect. Upon reduction, an important movement of the backbone region surrounding hemes I and II was also identified, that, together with a few redox-linked conformational changes in side-chain residues, results in a significant decrease in the solvent accessibility of hemes I and II.

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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.

Escherichia coli flavorubredoxin is a new type of cytoplasmic nitric oxide (NO) reductase, which shows NO reductase activity within the range of the canonical membrane-bound heme b 3 -iron NO reductases. Using reverse-transcription polymerase chain reaction we show that although the flavorubredoxin gene (flrd) is transcribed in both aerobic and anaerobic conditions, anaerobiosis induced transcription up to 12-fold, under fermentative conditions ; a 28-fold stimulation was observed in an E. coli fnr mutant strain, showing that the flavorubredoxin gene is negatively regulated by FNR. The level of anaerobic transcription was repressed three-fold by nitrate, but induced 47-fold by nitrite. The transcription factors NarL and NarP are not essential for flrd expression. Furthermore, the addition of NO within the physiological range of concentrations does not induce anaerobic transcription of flrd. Since two other E. coli proteins are known to exhibit NO reductase activity, flavohemoglobin and the pentaheme cytochrome c nitrite reductase, we have also compared the concentrations of their mRNAs with those of flavorubredoxin, under the same growth conditions. Transcription of the putative transcriptional activator of flavorubredoxin, ygaA, is also regulated by the absence of oxygen and the presence of nitrite. Levels of FlRd protein did not correlate with mRNA levels. The results reveal that a complex regulation of flavorubredoxin expression is operative, possibly by both transcriptional and post-transcriptional mechanisms. ß

Cytochromes c 3 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 c 3 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,. Threonine 24 was replaced with valine by sitedirected 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 redoxBohr 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.

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SummaryThe anaerobic bacterium Desulfovibrio desulphuricans ATCC 27774 contains a unique bacterioferritin, isolated with a stable di-iron centre and having ironcoproporphyrin III as its haem cofactor, as well as a type 2 rubredoxin with an unusual spacing of four amino acid residues between the first two binding cysteines. The genes encoding for these two proteins were cloned and sequenced. The deduced amino acid sequence of the bacterioferritin shows that it is among the most divergent members of this protein family. Most interestingly, the bacterioferritin and rubredoxin-2 genes form a dicistronic operon, which reflects the direct interaction between the two proteins. Indeed, bacterioferritin and rubredoxin-2 form a complex in vitro, as shown by the significant increase in the anisotropy and decay times of the fluorescence of rubredoxin-2 tryptophan(s) when mixed with bacterioferritin. In addition, rubredoxin-2 donates electrons to bacterioferritin. This is the first identification of an electron donor to a bacterioferritin and shows the involvement of rubredoxin-2 in iron metabolism. Furthermore, analysis of the genomic data for anaerobes suggests that rubredoxins play a general role in iron metabolism and oxygen detoxification in these prokaryotes.

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