Vectorial proton transfer coupled to reduction of O 2 and NO by a heme-copper oxidase

Abstract

The heme-copper oxidase (HCuO) superfamily consists of integral membrane proteins that catalyze the reduction of either oxygen or nitric oxide. The HCuOs that reduce O(2) to H(2)O couple this reaction to the generation of a transmembrane proton gradient by using electrons and protons from opposite sides of the membrane and by pumping protons from inside the cell or organelle to the outside. The bacterial NO-reductases (NOR) reduce NO to N(2)O (2NO + 2e(-) + 2H(+) --> N(2)O + H(2)O), a reaction as exergonic as that with O(2). Yet, in NOR both electrons and protons are taken from the outside periplasmic solution, thus not conserving the free energy available. The cbb(3)-type HCuOs catalyze reduction of both O(2) and NO. Here, we have investigated energy conservation in the Rhodobacter sphaeroides cbb(3) oxidase during reduction of either O(2) or NO. Whereas O(2) reduction is coupled to buildup of a substantial electrochemical gradient across the membrane, NO reduction is not. This means that although the cbb(3) oxidase has all of the structural elements for uptake of substrate protons from the inside, as well as for proton pumping, during NO reduction no pumping occurs and we suggest a scenario where substrate protons are derived from the outside solution. This would occur by a reversal of the proton pathway normally used for release of pumped protons. The consequences of our results for the general pumping mechanism in all HCuOs are discussed.