The diheme cytochrome c(4) from Vibrio cholerae is a natural electron donor to the respiratory cbb(3) oxygen reductase

Abstract

The respiratory chain of Vibrio cholerae contains three bd-type quinol oxygen reductases as well as one cbb(3) oxygen reductase. The cbb(3) oxygen reductase has been previously isolated and characterized; however, the natural mobile electron donor(s) that shuttles electrons between the bc(1) complex and the cbb(3) oxygen reductase is not known. The most likely candidates are the diheme cytochrome c(4) and monoheme cytochrome c(5), which have been previously shown to be present in the periplasm of aerobically grown cultures of V. cholerae. Both cytochromes c(4) and c(5) from V. cholerae have been cloned and expressed heterologously in Escherichia coli. It is shown that reduced cytochrome c(4) is a substrate for the purified cbb(3) oxygen reductase and can support steady state oxygen reductase activity of at least 300 e(-1)/s. In contrast, reduced cytochrome c(5) is not a good substrate for the cbb(3) oxygen reductase. Surprisingly, the dependence of the oxygen reductase activity on the concentration of cytochrome c(4) does not exhibit saturation. Global spectroscopic analysis of the time course of the oxidation of cytochrome c(4) indicates that the apparent lack of saturation is due to the strong dependence of K(M) and V(max) on the concentration of oxidized cytochrome c(4). 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 c(4) are 240 and 340 mV (vs standard hydrogen electrode), similar to the electrochemical properties of other c(4)-type cytochromes. Genomic analysis shows a strong correlation between the presence of a c(4)-type cytochrome and a cbb(3) oxygen reductase within the beta- and gamma-proteobacterial clades, suggesting that cytochrome c(4) is the likely natural electron donor to the cbb(3) oxygen reductases within these organisms. These would include the beta-proteobacteria Neisseria meningitidis and Neisseria gonnorhoeae, in which the cbb(3) oxygen reductases are the only terminal oxidases in their respiratory chains, and the gamma-proteobacterium Pseudomonas stutzeri.

Figure 1

SDS-PAGE of the isolated recombinant cytochromes c₄ and c₅. (A) 15% SDS gel stained with Coomassie blue. (B) Identical gel stained for heme. The lanes from left to right contain the cytochrome c₅ (lane 1), cytochrome c₄ (lane 2) and molecular weight standards (lane 3).

Figure 2

Absorption spectra of fully reduced (solid line) and fully oxidased (broken line) V. cholerae (A) cytochrome c₄ (3μM) and (B) cytochrome c₅ (7μM). The insets show the α- and β-bands of the fully reduced cytochrome c₄ (10μM) and cytochrome c₅ (17μM). All the absorption spectra were detected under the condition of 25 mM Tris-HCl (pH=7.5), 25 °C.

Figure 3

Cyclic voltammetry of the recombinant cytochrome c₄ which was applied to a freshly polished pyrolytic PGE electrode. The solution contained 10 mM HEPES buffer and 2 M NaCl at pH 7.0. Values obtained (vs. SHE) were E_pc1 = 0.26 V, E_pc2 = 0.35 V, E_pa1 = 0.23 V, E_pa2 = 0.34 V and E_1/2 = (E_pc + E_pa)/2 = 0.24 V and 0.34 V.

Figure 4

Steady state oxidase activity of the cbb₃ oxygen reductase from V. cholerae. (A) Dependence on the concentration of cytochrome c₄ (solid squares), cytochrome c₅ (open circles) and horse heart cytochrome c (solid triangles). The reaction mixture contained 50 mM sodium phosphate (pH 6.5), 50mM NaCl, 0.05% dodecylmaltoside, 10 mM sodium ascorbate, and the indicated amount of the cytochrome c, in a total volume of 1.8 ml. The O₂ consumption reaction was initiated by the addition of 50 nM of the oxidase. (B) Sensitivity of the oxidase activity to the addition of 25 μM cyanide. (C) Dependence of the oxidase activity on the concentration NaCl, measured with50 nM oxidase and 20 μM cytochrome c₄, in the presence of 10mM sodium ascorbate, 10 mM sodium phosphate (pH 6.5) buffer and 0.05% dodecylmaltoside.

Figure 5

The rates of oxidation of reduced cytochrome c₄ by the cbb₃ oxygen reductase from V. cholerae at different oxidized cytochrome c₄ concentrations determined by stopped-flow spectroscopy and least square fitting. The substrate [S] refers to reduced (not total) cytochrome c₄. The oxidized cytochrome c₄ concentrations were (A)1.5 μM; (B) 2.0 μM, (C) 2.5 μM; (D) 3.0 μM and (E) 7.5 μM. The curves show the non-linear least square fitting of the points to Michaelis-Menten kinetics. The K_M and V_max values for 1.5 μM, 2.0 μM and 2.5 μM oxidized cytochrome c₄ are plotted in (F).