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

Abstract

The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copper oxygen reductases (HCOs). Previous studies have established that C-family HCOs contain a single channel for uptake from the bacterial cytoplasm of all chemical and pumped protons, and that the entrance of the K C -channel is a conserved glutamate in subunit III. However, the majority of the K C -channel is within subunit I, and the pathway from this conserved glutamate to subunit I is not evident. In the present study, molecular dynamics simulations were used to characterize a chain of water molecules leading from the cytoplasmic solution, passing the conserved glutamate in subunit III and extending into subunit I. Formation of the water chain, which controls the delivery of protons to the K Cchannel, was found to depend on the conformation of Y241 Vc , located in subunit I at the interface with subunit III. Mutations of Y241 Vc (to A/F/H/S) in the Vibrio cholerae cbb 3 eliminate catalytic activity, but also cause perturbations that propagate over a 28-Å distance to the active site heme b 3 . The data suggest a linkage between residues lining the K C -channel and the active site of the enzyme, possibly mediated by transmembrane helix α7, which contains both Y241 Vc and the active site crosslinked Y255 Vc , as well as two Cu B histidine ligands. Other mutations of residues within or near helix α7 also perturb the active site, indicating that this helix is involved in modulation of the active site of the enzyme.oxygen reductase | proton pathway | bioenergetics | Vibrio cholerae | cbb 3

The two transmembrane helices of CcoP are sufficient for assembly of the cbb3-type heme-copper oxygen reductase from Vibrio cholerae

“…The K C -proton channel that is required to deliver protons to the active site for catalytic function has its entrance, E49, within the remaining hydrophobic domain of CcoP X (24, 25). Mutation of this residue (E49A) in the full-length V. cholerae cytochrome cbb 3 reduces the TMPD oxidase activity to 10% of the wild type value (24, 25).…”

“…Mutation of this residue (E49A) in the full-length V. cholerae cytochrome cbb 3 reduces the TMPD oxidase activity to 10% of the wild type value (24, 25). The same mutation, E49A, in the CcoNOQP X variant reduces the activity from 9% to less than 3% of the wild type activity (Table 1).…”

“…For the wild type enzyme, the results show that both hemes b and hemes c are oxidized in a single phase with time constant τ ≈ 0.3 ms ( k ≈ 3300 s −1 )(25). This is shown for both hemes b (Figure 9B and D) and hemes c (Figure 9A and C).…”

“…For the TMPD oxidase activity, the reaction mixture contained buffer (50 mM Sodium phosphate, 100 mM NaCl and 0.05% DDM at pH 6.5), 10 mM ascorbate and 500 μM TMPD as previously described (25). The pH dependence of the TMPD enzyme activity displays only data between pH 6.5 and pH 9.0 since below pH 6.5 the enzyme activity was decreased.…”

“…The enzyme samples were prepared as previously described (25). Briefly, the enzyme at a concentration of ∼5 μM was transferred to a modified anaerobic cuvette and the atmosphere was exchanged to N 2 on a vacuum line.…”