RESUMEN
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.
Asunto(s)
Grupo Citocromo c/química , Desulfovibrio desulfuricans/química , Modelos Moleculares , Aminoácidos/química , Cristalografía por Rayos X , Hemo/química , Concentración de Iones de Hidrógeno , Oxidación-Reducción , Conformación Proteica , Electricidad Estática , TermodinámicaRESUMEN
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.