A pathway for protons in nitric oxide reductase from Paracoccus denitrificans

Abstract

Nitric oxide reductase (NOR) from P. denitrificans is a membrane-bound protein complex that catalyses the reduction of NO to N(2)O (2NO+2e(-)+2H(+)-->N(2)O+H(2)O) as part of the denitrification process. Even though NO reduction is a highly exergonic reaction, and NOR belongs to the superfamily of O(2)-reducing, proton-pumping heme-copper oxidases (HCuOs), previous measurements have indicated that the reaction catalyzed by NOR is non-electrogenic, i.e. not contributing to the proton electrochemical gradient. Since electrons are provided by donors in the periplasm, this non-electrogenicity implies that the substrate protons are also taken up from the periplasm. Here, using direct measurements in liposome-reconstituted NOR during reduction of both NO and the alternative substrate O(2), we demonstrate that protons are indeed consumed from the 'outside'. First, multiple turnover reduction of O(2) resulted in an increase in pH on the outside of the NOR-vesicles. Second, comparison of electrical potential generation in NOR-liposomes during oxidation of the reduced enzyme by either NO or O(2) shows that the proton transfer signals are very similar for the two substrates proving the usefulness of O(2) as a model substrate for these studies. Last, optical measurements during single-turnover oxidation by O(2) show electron transfer coupled to proton uptake from outside the NOR-liposomes with a tau=15 ms, similar to results obtained for net proton uptake in solubilised NOR [U. Flock, N.J. Watmough, P. Adelroth, Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen, Biochemistry 44 (2005) 10711-10719]. NOR must thus contain a proton transfer pathway leading from the periplasmic surface into the active site. Using homology modeling with the structures of HCuOs as templates, we constructed a 3D model of the NorB catalytic subunit from P. denitrificans in order to search for such a pathway. A plausible pathway, consisting of conserved protonatable residues, is suggested.

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

“…Bacterial NOR is a membrane-bound protein that catalyzes one step of this process, namely, the two-electron reduction of NO to N 2 O (17,19). With no crystal or solution structure available for bacterial NOR to date, sequence alignments and homology modeling (21,22) have indicated that NOR is structurally homologous to the largest subunit (subunit I) of hemecopper oxidases (HCOs) (23), enzymes that catalyze reduction of O 2 to water. The active sites of both NOR and HCO contain a proximal histidine-coordinated heme and a distal three histidine-coordinated metal center.…”

“…However, the metal center in HCOs is occupied by a copper (called Cu B ), whereas a nonheme iron is present in NOR (called Fe B ) (23,24). 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).…”

“…The E122A, E125A, E198A, and E267A variants were inactive, indicating that these four glutamates are crucial for NOR activity (24,25,32,33). On the other hand, Reimann et al constructed a 3D model of NorB using homology modeling with the structures of HCOs as templates and suggested a plausible pathway consisting of these conserved glutamates for proton delivery (22). Despite these successes, the roles of the conserved glutamates and metal ions still remain to be fully elucidated, partly because of the difficulty in obtaining native NOR in high yield and the lack of a 3D structure.…”

Crystal structures of nitric oxide reductases provide key insights into functional conversion of respiratory enzymes

“…Contrary to COXs, the proton transfer pathway in NOR was less established, but biochemical and biophysical studies unambiguously showed no proton pumping activity and the catalytic proton transfer from the outside of the membrane in cNOR (11,12). The structure of P. aeruginosa cNOR demonstrated that there is no proton transfer pathway from the inside of the membrane and suggested two possible proton transfer pathways, channels 1 and 2, from the outside of the membrane (13) (Fig.…”

“…NORs and some COXs have crossreactivity with respective substrate (8)(9)(10), further supporting the view that NOR is evolutionary related to COX. However, NOR shows no proton pumping activity (11,12), whereas COX can generate the proton gradient by pumping protons in a process coupled with O 2 reduction. The produced proton gradient is essential for efficient ATP synthesis.…”