Mechanism for N₂O generation in bacterial nitric oxide reductase: a quantum chemical study.

The catalytic mechanism of reduction of NO to N(2)O in the bacterial enzyme nitric oxide reductase has been investigated using hybrid density functional theory and a model of the binuclear center (BNC) based on the newly determined crystal structure. The calculations strongly suggest a so-called cis:b(3) mechanism, while the commonly suggested trans mechanism is found to be energetically unfavorable. The mechanism suggested here involves a stable cis-hyponitrite, and it is shown that from this intermediate one N-O bond can be cleaved without the transfer of a proton or an electron into the binuclear active site, in agreement with experimental observations. The fully oxidized intermediate in the catalytic cycle and the resting form of the enzyme are suggested to have an oxo-bridged BNC with two high-spin ferric irons antiferromagnetically coupled. Both steps of reduction of the BNC after N(2)O formation are found to be pH-dependent, also in agreement with experiment. Finally, it is found that the oxo bridge in the oxidized BNC can react with NO to give nitrite, which explains the experimental observations that the fully oxidized enzyme reacts with NO, and most likely also the observed substrate inhibition at higher NO concentrations.