Abstract

The Escherichia coli cytochrome bo is a heme-copper terminal ubiquinol oxidase, and functions as a redox-driven proton pump. We applied pulse radiolysis technique for studying the one-electron reduction processes in the CuB-deficient mutant, His333Ala. We found that the CuB deficiency suppressed the heme b-to-heme o electron transfer two order of the magnitude (4.0×102 s–1), as found for the wild-type enzyme in the presence of 1 mM KCN (3.0×102 s–1). Potentiometric analysis of the His333Ala mutant revealed the 40 mV decrease in the Em value for low-spin heme b and the 160 mV increase in the Em value of high-spin heme o. Our results indicate that CuB not only serves as one-electron donor to the bound dioxygen upon the O-O bond cleavage, but also facilitates dioxygen reduction at the heme-copper binuclear centre by modulating the Em value of heme o through magnetic interactions. And the absence of a putative OH bound to CuB seems not to affect the uptake of the first chemical proton via K-channel in the His333Ala mutant.

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