Nitric Oxide Reduction by Heme‐Thiolate Enzymes (P450nor): A Reevaluation of the Mechanism

The details of the heme‐thiolate nitric oxide reductase (P450nor) catalytic mechanism are still controversial. One theory, supported by computational results [D. L. Harris, Int. J. Quantum Chem. 2002 , 88 , 183−200], assumes two sequential one‐electron transfers from NAD(P)H to an initial [FeNO] 6 complex. The [FeNO] 8 species thus formed would react with NO, eventually liberating the unstable ONNO 2− anion (most probably in its protonated form), which decomposes to N 2 O and water. However, more recent experimental results [A. Daiber et al., J. Inorg. Biochem. 2002 , 88 , 343−352] suggest the first committed step of the mechanism to be direct hydride transfer from NAD(P)H to [FeNO] 6 , presumably resulting in an iron‐bound HNO unit, [Fe‐(H)NO] 8 , that would be readily protonated to [Fe‐(H)NOH] 8 . Subsequent NO addition would yield the unstable HO‐N(H)‐N=O, which would dissociate from the heme and decompose to H 2 O and N 2 O. Here, the DFT geometry optimization of all previously proposed reaction intermediates is reported. The first step of the mechanism is predicted to be hydride transfer to [FeNO] 6 , to produce [FeNOH] 8 or [Fe‐N(H)O] 8 . Subsequent addition of NO to [Fe‐NOH] 8 (but not to [Fe‐N(H)O] 8 or [Fe‐N(H)OH] 8 ) is predicted to lead to immediate liberation of HN 2 O 2 − , without any stable intermediates. Contrary to what would be predicted according to the “thiolate push effect” dogma, the thiolate ligand at the heme active site is shown to obstruct NO reduction , rather than facilitate it. It is in fact shown that replacement of the thiolate by a neutral nitrogen ligand (i.e., lysine, as found in the active site of cytochrome c nitrite reductase, an enzyme that can reduce NO) clearly favors, from a thermodynamic point of view, NO reduction at the heme site. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)