Identification of nitric oxide reductase activity in Rhodobacter capsulatus: the electron transport pathway can either use or bypass both cytochrome c2 and the cytochrome bc1 complex

Abstract

Several strains of Rhohbaeter capsufatus have been shown to possess a nitric oxide reductase activity (reaction product nitrous oxide) after anaerobic phototrophic growth, but not after aerobic growth. The reductase is associated with the cytoplasmic membrane and electrons can reach the enzyme via the cytochrome bel complex. However, use of appropriate strains has shown that neither the latter, cytochrome c2 nor cytochrome c' is essential for the reduction of nitric oxide. Inhibition by myxothiazol of nitric oxide reduction in a strain that lacks a cytochrome c2 establishes that in phototrophically grown R. capsufatus the cytochrome bel complex is able to transfer electrons to an acceptor that is alternative to cytochrome c2. Electron transport to nitric oxide from NADH or succinate generated a membrane potential. When isoascorbate plus 2,3,5,6-tetramethyl-p-phenylenediamine (DAD) was the electron donor a membrane potential was not generated. This observation implies that nitric oxide is reduced at the periplasmic surface of the membrane and that the reductase is not proton translocating.

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

“…In cNOR, even though NO reduction is similarly exergonic to O 2 reduction, NORs do not pump protons, and substrate protons are taken from the periplasmic solution (19,20,40), as supported also by the recent crystal structure of cNOR from Pseudomonas aeruginosa, showing several possible proton pathways from the periplasm but none from the cytoplasm (41). The cbb 3 oxidases from R. sphaeroides and P. stutzeri have been found to catalyze NO reduction (9, 10), and we recently investigated the coupling of NO reduction to proton translocation in the cbb 3 s by using the flow-flash technique in combination with both optical and electrometric detection in liposome-reconstituted cbb 3 (10).…”

The Nitric-oxide Reductase from Paracoccus denitrificans Uses a Single Specific Proton Pathway

“…1 and 2). cNOR, on the other hand, has been shown not to contribute to the electrochemical proton gradient, although the amount of free energy available from NO reduction is similar to that for O 2 reduction (3)(4)(5). Electrons are donated by soluble carriers in the periplasm, so for the overall reaction to be non-electrogenic, protons used for NO reduction must also originate in the periplasmic (outside) solution.…”

“…However, the two loops with Gly-340 and Gly-69 C together with the Tyr-73 C (which ligates a Ca 2ϩ ; see Fig. 1) prevented water from this cavity reaching the water cluster around the heme b 3 propionates further on in the path. Pathway 2 was therefore concluded to be unlikely (15), but it could not be excluded without experimental data.…”

“…NO reduction takes place in the NorB subunit that contains three redox centers: two b-type hemes (hemes b and b 3 ) and a non-heme iron (Fe B ). Heme b 3 and the non-heme iron form the binuclear center, where nitric oxide is bound and reduced. NorC is a membrane-anchored cytochrome c with one c-type heme, which presumably forms the site of electron entry.…”

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

“…In significant contrast to the O 2 -reducing HCuOs, NORs do not couple the energetically downhill reduction of NO, which is as exergonic as O 2 reduction, to the generation of a proton gradient across the membrane. NO reduction by NOR is thus nonelectrogenic (14,16,17) and protons are taken up from the outside (periplasmic) solution (14). Different reasons are possible for the lack of coupling of NO reduction to the generation of a proton gradient.…”