Vectorial proton transfer coupled to reduction of O2 and NO by a heme-copper oxidase

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.