NO Reduction by Nitric-oxide Reductase from Denitrifying Bacterium Pseudomonas aeruginosa

Abstract

Nitric-oxide reductase (NOR) of a denitrifying bacterium catalyzes NO reduction to N 2 O at the binuclear catalytic center consisting of high spin heme b 3 and non-heme Fe B . The structures of the reaction intermediates in the single turnover of the NO reduction by NOR from Pseudomonas aeruginosa were investigated using optical absorption and EPR spectroscopies combined with an originally designed freeze-quench device. In the EPR spectrum of the sample, in which the fully reduced NOR was mixed with an NO solution and quenched at 0.5 ms after the mixing, two characteristic signals for the ferrous Fe B -NO and the penta-coordinated ferrous heme b 3 -NO species were observed. The CO inhibition of its formation indicated that two NO molecules were simultaneously distributed into the two irons of the same binuclear center of the enzyme in this state. The time-and temperature-dependent EPR spectral changes indicated that the species that appeared at 0.5 ms is a transient reaction intermediate prior to the N 2 O formation, in good agreement with the so-called "trans" mechanism. It was also found that the final state of the enzyme in the single turnover cycle is the fully oxidized state, in which the -oxo-bridged ligand is absent between the two irons of its binuclear center, unlike the resting form of NOR as isolated. On the basis of these present findings, we propose a newly developed mechanism for the NO reduction reaction conducted by NOR. Nitric-oxide reductase (NOR)1 is a metal-containing enzyme involved in biological denitrification processes. The enzyme catalyzes the two-electron reduction of nitric oxide (NO) to nitrous oxide (N 2 O) according to Equation 1 (1-4). Since the NO reduction reaction involves the N-O bond cleavage and the N-N bond formation, it is chemically interesting to elucidate the molecular mechanism of the NO reduction catalyzed by the metal enzymes.

Substrate Control of Internal Electron Transfer in Bacterial Nitric-oxide Reductase

“…This means that reaction schemes involving such electron redistribution before NO binding (such as in Ref. 19) are incompatible with our data. A simplified reaction scheme consistent with the data obtained in this study is shown in Fig.…”

“…It should be noted that we do not expect the binding of NO to Fe B to give rise to any significant absorbance changes at the wavelengths used, so Fe B might bind NO faster than the heme b 3 , or it could bind NO during the 200-ms mixing time, with CO still bound to the heme b 3 . Such simultaneous binding of CO to heme b 3 and NO to Fe B has been suggested previously (19). The second NO molecule could also bind to the intermediate formed at heme b 3 upon binding of the first (21).…”

“…The detailed mechanism for NO reduction by NOR is not known, but models have been suggested that involve either a "trans" mechanism (16,18,19), where one NO binds to each of the metals in the binuclear site, or "cis" mechanisms, where two NOs bind either to the non-heme Fe B (20; see also 14), or consecutively to heme b 3 such that the second NO binds to the intermediate formed upon binding of the first (21).…”

Spectroscopic characterization of heme iron–nitrosyl species and their role in NO reductase mechanisms in diiron proteins

“…58 One signal, at g = 4, is consistent with a non-heme S = 3/2 {FeNO} 7 species, and another at g = 2.01, with a pronounced 3-line hyperfine structure, is characteristic of a heme S = 1/2 {FeNO} 7 system. Shiro and coworkers estimate that these two nitrosyl signals represent 30% of the total diiron site concentration.…”

“…57 Also, rapid-freeze quench samples from single turnover reactions in cNOR have revealed EPR signals consistent with the trapping of S = 1/2 low-spin heme {FeNO} 7 and S = 3/2 non-heme {FeNO} 7 species. 58 …”