Mechanism of Nitric Oxide Reduction at Biological Heme Active Sites

Nitric oxide (NO) plays pivotal roles in various biological processes although it shows high cytotoxicity. In microbial denitrification, in which nitrate is reduced to dinitrogen in a step-wise manner, NO is produced as an intermediate product. To eliminate the cytotoxic effect of NO, microorganisms utilize NO reductase (NOR), which decomposes two NO molecules to nitrous oxide (N2O) (2NO + 2H+ + 2e- → N2O + H2O). To understand the mechanism of NO reduction, we have designed the time-resolved (TR) techniques using caged NO, which produces NO upon UV illumination, as a reaction trigger. The validity of the TR techniques with caged NO was demonstrated by the studies on the reaction mechanism of soluble NOR in which a thiolate-coordinated heme works as an active center [1-2]. Here, we tried to further develop the method using caged NO and applied this technique to elucidate the reaction mechanism of membrane-integrated NOR from Pseudomonas aeruginosa (cNOR). cNOR catalyzes reductive coupling of two NO molecules to nitrous oxide (N2O) using two protons and reducing equivalents at a heme/non-heme Fe binuclear center. Human pathogens such as P. aeruginosa utilize cNOR to decompose NO produced from hosts’ immune system, indicating the biological importance of cNOR. In addition, because this reaction contains fundamental elements for chemical reactions such as the N-N bond formation and the N-O bond cleavage, the reaction mechanism will provide how the metal center effectively proceeds the chemical reaction. To follow the catalytic reaction of cNOR, the TR-visible absorption spectra for cNOR were measured using caged NO as a reaction trigger and a source of the substrate. The TR data indicated the following mechanism [3]. One NO molecule binds to reduced cNOR to form intermediate 1 in ~5 µs, followed by the formation of intermediate 2 without protonation and second NO biding at ~100 µs. Finally, the binding of second NO and the protonation to intermediate 2 yield N2O in ~ms time region. To get further insights into the structure of the intermediates, we aimed to trap the reaction intermediates by the photolysis of caged NO under cryogenic temperature (cryo-photolysis) and following thermal annealing. The cryo-photolysis of caged NO with reduced cNOR and subsequent annealing at ~160 K produced a species showing an EPR signal at g = ~4.0, which is assignable to non-heme Fe-NO species [4]. Because the NO stretching mode was detected at the region of non-heme NO species, 1683 cm-1, by the TR-IR measurement with caged NO at 5 µs, intermediate 1 is a non-heme Fe-NO species [4]. On the basis of the data obtained by the method using caged NO, more details on the catalytic mechanism in cNOR will be discussed. REFERENCES Tosha et al. Nat. Commun., 2017, 8, 1584. Nomura et al. Proc. Natl. Acad. Sci. USA, 2021, 118, e2101481118. Takeda et al. Bull. Chem. Soc. Jpn. 2020, 93, 825-833. Takeda et al. J. Phys. Chem. B, 2023, 127, 846-854.