The bacterial respiratory nitric oxide reductase

Abstract

The two-subunit cytochrome bc complex (NorBC) isolated from membranes of the model denitrifying soil bacterium Paracoccus denitrificans is the best-characterized example of the bacterial respiratory nitric oxide reductases. These are members of the super-family of haem-copper oxidases and are characterized by the elemental composition of their active site, which contains non-haem iron rather than copper, at which the reductive coupling of two molecules of nitric oxide to form nitrous oxide is catalysed. The reaction requires the presence of two substrate molecules at the active site along with the controlled input of two electrons and two protons from the same side of the membrane. In the present paper, we consider progress towards understanding the pathways of electron and proton transfer in NOR and how this information can be integrated with evidence for the likely modes of substrate binding at the active site to propose a revised and experimentally testable reaction mechanism.

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

“…Although two conserved glutamates (E198 and E267) are known to be crucial for NOR activity (24,25), their roles are not well defined (18,19). In a previous study (14), we demonstrated that one Glu, E68, is important for both iron binding and NOR activity of Fe B Mb.…”

“…3A). Since deoxy myoglobin contains Fe(II) heme that exhibits no EPR signals in X-band EPR (14), we added blue copper Cu(II)-azurin (49), a redox partner of native NOR (19), to oxidize both the reduced heme and nonheme irons in Fe (II)-I107E Fe B Mb to EPR-active Fe(III). Upon addition of Cu (II)-azurin, the oxidation of deoxy I107E Fe B Mb resulted in EPR signals at g ¼ 6.12 and 5.56, typical of a high-spin hemeFe(III).…”

“…Bacterial denitrification is a crucial part of the nitrogen cycle in nature that involves a four-step, five-electron reduction of nitrate (NO 3 − ) to dinitrogen (N 2 ) (17,19). 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).…”

“…Bacterial denitrification is a crucial part of the nitrogen cycle in nature that involves a four-step, five-electron reduction of nitrate (NO 3 − ) to dinitrogen (N 2 ) (17,19). 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.…”

“…While being able to design such functional proteins is laudable, the impact of such an achievement would be greater if the designed proteins can be used to address fundamental issues in chemistry and biology that are difficult to tackle by other methods. One primary example is the roles of conserved glutamates and metal ions in bacterial nitric oxide reductase (NOR) (17)(18)(19).NO is critical for all life (20). Bacterial denitrification is a crucial part of the nitrogen cycle in nature that involves a four-step, five-electron reduction of nitrate (NO 3 − ) to dinitrogen (N 2 ) (17, 19).…”