The Ubiquinol Binding Site of Cytochrome from Accommodates Menaquinone and Stabilizes a Functional Menasemiquinone.

Cytochrome , one of three terminal oxygen reductases in the aerobic respiratory chain of , has been well characterized as a ubiquinol oxidase. The ability of cytochrome to catalyze the two-electron oxidation of ubiquinol-8 requires the enzyme to stabilize the one-electron oxidized ubisemiquinone species that is a transient intermediate in the reaction. Cytochrome has been shown recently to also utilize demethylmenaquinol-8 as a substrate that, along with menaquinol-8, replaces ubiquinol-8 when is grown under microaerobic or anaerobic conditions. In this work, we show that its steady-state turnover with 2,3-dimethyl-1,4-naphthoquinol, a water-soluble menaquinol analogue, is just as efficient as with ubiquinol-1. Using pulsed electron paramagnetic resonance spectroscopy, we demonstrate that the same residues in cytochrome that stabilize the semiquinone state of ubiquinone also stabilize the semiquinone state of menaquinone, with the hydrogen bond strengths and the distribution of unpaired spin density accommodated for the different substrate. Catalytic function with menaquinol is more tolerant of mutations at the active site than with ubiquinol. A mutation of one of the stabilizing residues (R71H in subunit I) that eliminates the ubiquinol oxidase activity of cytochrome does not abolish activity with soluble menaquinol analogues.