Partial Steps of Charge Translocation in the Non-Pumping Mutant N139L of Rhodobacter Sphaeroides Cytochrome C Oxidase with a Blocked D-Channel

摘要

N139L substitution in D-channel of cytochrome oxidase from Rhodobacter sphaeroides results in a ∼15-fold decrease of turnover number and in loss of proton pumping. Time-resolved absorption and electrometric assays of the >F→O transition in the N139L mutant oxidase result in 3 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 the enzyme turnover (∼30 s^-1). Presumably, an abnormal intermediate >Fdeprotonated 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, the protonic phase of Δψ-generation coupled to the flash-induced >F→O transition greatly decreases in rate and magnitude and can be assigned to proton movement from E286 to the binuclear site, required for reduction of heme a3 from Fe⁴⁺=O^2- to 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, the KCN-insensitive rapid electrogenic phase may be actually composed of two components with lifetimes of ∼10 and ∼40 μs and the magnitude ratio of ∼3:2, respectively. The 10 μs phase matches vectorial electron transfer from CuA 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 2 charges per electron.