Formation of a cytochrome c–nitrous oxide reductase complex is obligatory for N2O reduction by Paracoccus pantotrophus

Abstract

Nitrous oxide reductase (N 2 OR) catalyses the final step of bacterial denitrification, the two-electron reduction of nitrous oxide (N 2 O) to dinitrogen (N 2 ). N 2 OR contains two metal centers; a binuclear copper center, Cu A , that serves to receive electrons from soluble donors, and a tetranuclear copper-sulfide center, Cu Z , at the active site. Stopped flow experiments at low ionic strengths reveal rapid electron transfer (k obs = 150 s −1 ) between reduced horse heart (HH) cytochrome c and the Cu A center in fully oxidized N 2 OR. When fully reduced N 2 OR was mixed with oxidized cytochrome c, a similar rate of electron transfer was recorded for the reverse reaction, followed by a much slower internal electron transfer from Cu Z to Cu A (k obs = 0.1-0.4 s −1 ). The internal electron transfer process is likely to represent the rate-determining step in the catalytic cycle. Remarkably, in the absence of cytochrome c, fully reduced N 2 OR is inert towards its substrate, even though sufficient electrons are stored to initiate a single turnover. However, in the presence of reduced cytochrome c and N 2 O, a single turnover occurs after a lag-phase. We propose that a conformational change in N 2 OR is induced by its specific interaction with cytochrome c that in turn either permits electron transfer between Cu A and Cu Z or controls the rate of N 2 O decomposition at the active site.

The tetranuclear copper active site of nitrous oxide reductase: the CuZ center

“…Small electron donor proteins, as c-type cytochromes, either physiological or nonphysiological (mitochondrial cytochrome c), have been used as electron donors to N 2 OR in in vitro assays [81]. In the case of N 2 OR isolated from Rhodobacter capsulatus, Rhodobacter sphaeroides f. sp.…”

“…In the case of N 2 OR isolated from Rhodobacter capsulatus, Rhodobacter sphaeroides f. sp. denitrificans, and Paracoccus pantotrophus [37,82,83], the physiological donor is a periplasmic c-type cytochrome, but Paracoccus pantotrophus N 2 OR can also accept electrons from a periplasmic type 1 copper protein, pseudoazurin [37], and from the mitochondrial horse heart cytochrome c [81]. Bovine heart cytochrome c is able to reduce Achromobacter cycloclastes N 2 OR [84], but its physiological electron donor is pseudoazurin [85].…”

The electron transfer complex between nitrous oxide reductase and its electron donors

“…This high homology justifies not only the use of the P. panthotropus pseudoazurin structure in the docking studies, but also the use of the biochemical properties of P. panthotropus N 2 OR. It was reported that the increase in ionic strength decreases the activity of P. panthotropus N 2 OR in the presence of horse heart cytochrome c, which is an indication that the complex formed has an electrostatic nature [22]. The Fig.…”

“…Previously, direct electron transfer studies have shown for P. panthotropus and P. nautica N 2 OR that the small electron transfer proteins donate electrons directly to the binuclear CuA center of N 2 OR [18,22]. Therefore, the solutions were filtered using the condition that the distance between the redox centers, the CuA center and the iron ion of cytochrome c, or the copper ion of pseudoazurin, should be less than 20 Å , a condition required for an efficient electron transfer [15] (Table 1, and their representation is shown in Figs.…”

“…A steady-state kinetic study demonstrated that the interaction between the two proteins is mainly hydrophobic in nature and that mitochondrial cytochrome c is not a competent electron donor to this enzyme [18]. On the other hand, N 2 OR isolated from Paracoccus pantotrophus, an organism closely related to P. denitrificans, can accept electrons from cytochrome c-550 and pseudoazurin [19][20][21], and also from the mitochondrial cytochrome c [22]. A structural model for the electron transfer complex between P. denitrificans N 2 OR and either P. panthotropus cytochrome c-550 or P. panthotropus pseudoazurin has been proposed on the basis of a theoretical docking study [23].…”