The Diheme Cytochrome c₄ from Vibrio cholerae Is a Natural Electron Donor to the Respiratory cbb₃ Oxygen Reductase

Abstract

The respiratory chain of Vibrio cholerae contains three bd-type quinol oxygen reductases as well as one cbb3 oxygen reductase. The cbb3 oxygen reductase has been previously isolated and characterized, however the natural mobile electron donor(s) which shuttles electrons between the bc1 complex and the cbb3 oxygen reductase is not known. The most likely candidates are the diheme cytochrome c4 and mono-heme cytochrome c5, which have been previously shown to be present in the periplasm of aerobically grown cultures of V. cholerae. Both cytochromes c4 and c5 from V. cholerae have been cloned and expressed heterologously in E. coli. It is shown that reduced cytochrome c4 is a substrate for the purified cbb3 oxygen reductase and can support steady state oxygen reductase activity of at least 300 e−1/s. In contrast, reduced cytochrome c5 is not a good substrate for the cbb3 oxygen reductase. Surprisingly, the dependence of the oxygen reductase activity on the concentration of cytochrome c4 does not exhibit saturation. Global spectroscopic analysis of the time course of the oxidation of cytochrome c4 indicates that the apparent lack of saturation is due to the strong dependence of KM and Vmax on the concentration of oxidized cytochrome c4. Whether this is an artifact of the in vitro assay or has physiological significance remains unknown. Cyclic voltammetry was used to determine that the midpoint potentials of the two hemes in cytochrome c4 are 240 mV and 340 mV (vs SHE), similar to the electrochemical properties of other c4-type cytochromes. Genomic analysis shows a strong correlation between the presence of a c4-type cytochrome and a cbb3 oxygen reductase within the β- and γ- proteobacterial clades, suggesting that cytochrome c4 is the likely natural electron donor to the cbb3 oxygen reductases within these organisms. These would include the β-proteobacteria Neisseria meningitidis and Neisseria gonnorhoeae, in which the cbb3 oxygen reductases are the only terminal oxidases in their respiratory chains, and the γ- proteobacterium Pseudomonas stutzeri.

Conformational coupling between the active site and residues within the K C -channel of the Vibrio cholerae cbb 3 -type (C-family) oxygen reductase

“…The reaction mixture contained 1.8 mL of 50 mM sodium phosphate, 100 mM NaCl, and 0.05% dodecyl β-d-maltoside (DDM) at pH 6.5. Here 500 μM N,N,N′,N′-tetramethylp-phenylenediamine or 40 μM V. cholerae cytochrome c 4 , which is a natural electron donor for the V. cholerae cytochrome cbb 3 (40), with 10 mM ascorbate, was used as a reductant for enzyme activity assays at 25°C.…”

Electrochemical study of an electron shuttle diheme protein: The cytochrome c from T. thermophilus

“…A unique pair of quasi reversible anodic and cathodic signals was observed for the three protein samples. Again, this is different from cyt c4 which exhibited four well-defined redox peaks, two in oxidation and two in reduction [31,34]. The redox potentials, determined from the half sum of the peak potentials, are +0.14 V for both the full-length protein and the C-terminal fragment and +0.…”

“…The similar redox potential of both hemes and the overlap of their spectral contributions in the -band at 550 nm do not allow to distinguish them clearly in the UV/Vis potentiometric titration. In contrast, the two hemes of cyt c4 have significantly different midpoint potentials, one above +300 mV and the second between +190 and +260 mV [31][32][33][34]. This leads to more complexity in the redox titration [32].…”

“…Mainly hydrophobic interactions are thus believed to take place between cyt c552 and the corresponding cyt c oxidases in T. thermophilus [25,28,29]. Bacteria also use diheme proteins to transfer electrons between the respiratory enzyme complexes, such as the cyt c4, which are native electron donors of C family (cbb3) oxidases [30,31]. They are the simplest model systems for multi heme proteins [31][32][33][34].…”

“…Bacteria also use diheme proteins to transfer electrons between the respiratory enzyme complexes, such as the cyt c4, which are native electron donors of C family (cbb3) oxidases [30,31]. They are the simplest model systems for multi heme proteins [31][32][33][34]. Recently, a diheme electron carrier protein with 15% sequence identity only to cyt c4 was identified from T. thermophilus [35].…”