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 H2O 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 N2O (2NO ؉ 2e ؊ ؉ 2H ؉ 3 N2O ؉ H2O), a reaction as exergonic as that with O2. 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 O2 and NO. Here, we have investigated energy conservation in the Rhodobacter sphaeroides cbb3 oxidase during reduction of either O2 or NO. Whereas O2 reduction is coupled to buildup of a substantial electrochemical gradient across the membrane, NO reduction is not. This means that although the cbb3 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.cbb3 ͉ flow-flash ͉ nitric oxide ͉ proton pumping ͉ pathway

Entrance of the proton pathway in cbb 3 -type heme-copper oxidases

“…We previously described the reaction between fully reduced wild-type R. sphaeroides cbb 3 reconstituted into vesicles and O 2 (10), where the reaction is characterized by a major oxidation phase (of all heme groups) with a time constant ðτÞ ¼ 1 ms. In this work the solubilized cbb 3 was studied, and the O 2 concentration was 1 mM instead of 0.6 mM in the previous study.…”

“…Among the O 2 -reducing HCuOs, the cbb 3 type is the closest relative to NOR, and interestingly, cbb 3 -type oxidases, in contrast to A-type, have substantial NO-reduction activities (9,10). The A-type oxidases contain, in their catalytic subunit I, a lowspin heme a and a high-spin heme a 3 -Cu B active site.…”

“…S2 and S3) have hampered detailed kinetic studies using the flowflash technique. From our previous study (10), it is nevertheless clear that flow-flash studies are possible, and the fully reduced wild-type cbb 3 reacts with O 2 , all redox-active groups oxidize and protons are taken up in a reaction with a time constant of approximately 1 ms. Furthermore, our results indicated that when reducing NO, the cbb 3 , like NOR, uses protons from the periplasmic side of the membrane (10).…”

Roles of glutamates and metal ions in a rationally designed nitric oxide reductase based on myoglobin

“…The hydrogen binding network in our protein models may contribute to the fine-tuning of the Glu pK a to be more neutral, similar to that in NOR. Moreover, it is interesting that Cu(I)-I107E Fe B Mb also shows NOR activity, which provides an interesting protein model of HCOs with NOR function (26)(27)(28), even though Glu residues are not conserved in native HCOs. Additionally, spectroelectrochemical studies showed that the reduction potential of I107E Fe B Mb with no metal ion in the Fe B site is similar to that of Fe B Mb, but much lower than that of Cu B Mb (77 mV) (54), which contains the same three His, but no Glu in the metal-binding site above the heme.…”

“…In addition, two conserved glutamates, shown by modeling to be close to the Fe B site (21,22), are found to be essential for NOR activity (24,25). Some members of HCOs such as cytochrome cbb 3 oxidase display NOR activity (26)(27)(28), although the activity is ∼50-fold lower than native NOR (26). Therefore, it is important to elucidate the structural features, specifically the roles of the conserved glutamates close to the Fe B site and metal ions (copper vs. iron), responsible for the reduction of NO to N 2 O.…”