Partial steps of charge translocation in the nonpumping N139l mutant of rhodobacter sphaeroides cytochrome c oxidase with a blocked d-channel

The N139L substitution in the D-channel of cytochrome oxidase from Rhodobacter sphaeroides results in an ?15-fold decrease in the turnover number and a loss of proton pumping. Time-resolved absorption and electrometric assays of the F → O transition in the N139L mutant oxidase result in three major findings. (1) Oxidation of the reduced enzyme by O₂ shows ∼ 200-fold inhibition of the F → O step (k ∼ 2 s^-1 at pH 8) which is not compatible with enzyme turnover (∼30 s^-1). Presumably, an abnormal intermediate F_deprotonated is formed under these conditions, one proton-deficient relative to a normal F state. In contrast, the F → O transition in N139L oxidase induced by single-electron photoreduction of intermediate F, generated by reaction of the oxidized enzyme with H₂O₂, decelerates to an extent compatible with enzyme turnover. (2) In the N139L mutant, the protonic phase of Δ Ψ generation coupled to the flash-induced F → O transition greatly decreases in rate and magnitude and can be assigned to the movement of a proton from E286 to the binuclear site, required for reduction of heme a₃ from the Fe⁴⁺ - O^2- state to the Fe³⁺-OH^- state. Electrogenic reprotonation of E286 from the inner aqueous phase is missing from the F → O step in the mutant. (3) In the N139L mutant, the KCN-insensitive rapid electrogenic phase may be composed of two components with lifetimes of ∼ 10 and ∼ 40 μs and a magnitude ratio of ∼ 3:2. The 10 μ s phase matches vectorial electron transfer from Cu_A to heme a, whereas the 40 μs component is assigned to intraprotein proton displacement across -20% of the membrane dielectric thickness. This proton displacement might be triggered by rotation of the charged K362 side chain coupled to heme a reduction. The two components of the rapid electrogenic phase have been resolved subsequently with other D-channel mutants as well as with cyanide-inhibited wild-type oxidase. The finding helps to reconcile the unusually high relative contribution of the microsecond electrogenic phase in the bacterial enzyme (∼ 30%) with the net electrogenicity of the F → O transition coupled to transmembrane transfer of two charges per electron.