Evidence for an NO‐rebound, mechanism for production of N₂O from nitrite by the copper‐containing nitrite reductase from Achromobacter cycloclastes

Abstract

Reduction of NO; by the C&containing nitrite reductase from Achromobacfer cycloclustes produces NO as the primary product initially, but as NO accumulates, NO production levels-off and N,O production becomes significant. Reaction of the enzyme with NO; in the presence of NO increases the amount of NzO product significantly, while trapping the NO product as nitrosylhemoglobin or rapid. removal of NO by spaiging results in no detectable N20 production. Reaction of the enzyme with "NO; in the presence of 14N0 results in rapid formation of the mixed isotope product (idId, "N)O in ca. 45% yield. In contrast, the presence or absence of NO has no effect on N,O production by a prototypical heme &,-containing nitrite reductase. These results are consistent with formation of a labile Cu'-NO" species in the copper enzyme, which normally decomposes to NO. Production of N,O requires that the released NO must rebind to the enzyme to combine with a second NO; or a species derived therefrom.Denitrification; Nitrite reductase; Nitrosyl; Isotope labelling; Reaction mechanism; N1O production INTRODUCTIONDespite many detailed studies on intact bacteria, crude extracts, and purified enzymes [l-4], the mechanism of denitrification continues to be the object of controversy. Although it is now generally accepted that denitrifying bacteria possess a distinct nitric oxide reductase (NOR) activity and that at least a significant portion of the total nitrogen flux occurs via a stepwise pathway with NO as an intermediate (Eqn, 1), the importance of an alternative direct pathway in which 2 nitrite ions are reduced to N20 on a single enzyme, nitrite reductase (NiR), (Eqn. 2) remains unclear.On the one hand, quantitative studies of NO levels during denitrification by several organisms have been interpreted as indicating that only the stepwise pathway of Eqn. (1) NO [12], indicating that, for some organisms at least, NO is not an obligatory intermediate. This conclusion is supported by a number of isotope labelling and trapping studies [ 13-161, by the fact that the "N isotope effect increases with increasing nitrite concentration, suggesting that 2 nitrite ions combine prior to the first irreversible step [ 17,181, and by the fact that purified NiR's do in at least some cases produce significant amounts of N20 that cannot be attributed to chemical side reactions [19-211. In order to determine the origin of some of these apparently contradictory findings, we have examined the reduction of NO, by both the Cu-and heme cd,-containing NiR's in some detail. The problem is complicated by the fact that one of the products, NO, is known to be a potent inhibitor of NO; reduction by both types of enzyme [22,23]. In this communication, WC prcscnt evidence that NO binding to the Cu NiR of Volume 291, number 1 FEBS LETTERSOctober 199 1Achrornobacter cycioclastes is essential for N20 production by the enzyme. r EXPERIMENTALNiteite reductases were isolated from A. cyclocl~srcs [24] and Pseudomonos stutzeri [ZS] as peeviously described and stored frozen un...

Structures of a Blue-Copper Nitrite Reductase and its Substrate-Bound Complex

“…It has been suggested that the reaction of NO, generated during turnover as a product of the reduction of nitrite, reacts with the putative copper-nitrosyl intermediate (Jackson, Tiedje & Averill, 1991) to form N20. In the case of AcNiR the putative E--NO + has been trapped in the presence of NH2OH, and the production of N20 has been demonstrated via this intermediate (Hulse et AI45 ~ Fig.…”

Dissimilatory Nitrite and Nitric Oxide Reductases

“…94 Second, unlike the heme cd 1 enzymes, the Cu NiRs produce a significant amount of N 2 O from NO 2 -, but only after substantial levels of NO have built up. 101 Third, reaction of 15 …”

Catalytic Roles for Two Water Bridged Residues (Asp-98 and His-255) in the Active Site of Copper-containing Nitrite Reductase

“…The catalytic function of NIR is to catalyze the one electron reduction of nitrite (NO 2 Ϫ ) to nitric oxide (NO) under anaerobic conditions (3). In vitro, N 2 O is also produced from the close homologue Achromobacter cycloclastes NIR (AcNIR), if NO is not removed from the reaction (4). The in vivo electron donor to A. faecalis S-6 NIR (AfNIR) is pseudoazurin, a small blue copper protein that belongs to the cupredoxin family of electron transfer proteins (3,5).…”