Timing of NO Binding and Protonation in the Catalytic Reaction of Bacterial Nitric Oxide Reductase as Established by Time-Resolved Spectroscopy.

Membrane-integrated nitric oxide reductases (NOR) catalyze the formation of nitrous oxide (N2O) from two NO molecules using two protons and two electrons at a heme/non-heme iron binuclear center. Despite extensive efforts, the mechanism underlying the NOR-catalyzed reaction has been poorly understood due to the rapidity of the reaction. Here, we utilized a photosensitive caged NO compound as a trigger for the NOR reaction to characterize the NO reduction mechanism by time-resolved visible absorption spectroscopy. We showed that the NOR reaction consists of three steps. One NO molecule binds to the reduced binuclear center to form a non-heme Fe(II)-NO species in the 1st phase (microsecond timescale), followed by a migration of NO to form the other chemical species, possibly 5-coordinate heme b(3)-NO, in the 2nd phase (timescale of tens of microseconds). Then, the NO bound to heme reacts with a second NO molecule in the 3rd phase (millisecond timescale), in which protonation and electron transfer promote N-N bond formation and N-O bond cleavage to yield N2O. These findings led us to propose a revised trans mechanism for NO reduction by NOR.