Structural characterization of the catalytic high-spin heme b of nitric oxide reductase: A resonance Raman study

Nitric oxide reductase (NOR) from Paracoccus denitrificans is a transmembrane heterodimer containing a low-spin heme c, a low-spin heme b, a high-spin heme b, and a non-heme iron. Protein sequence similarities between NOR and the cytochrome oxidase superfamily suggest the catalytic center of NO reduction to be the dinuclear high-spin heme b/non-heme iron site and the two low-spin hemes to facilitate electron transfer. The EPR-silent character of the non-heme iron and the ferric high-spin heme b is believed to be due to an antiferromagnetic coupling between these two metal centers via a bridging ligand. Soret or red excitations on the fully reduced, reduced CO-bound, and fully oxidized states of NOR allow enhancement of the resonance Raman (RR) contributions of the catalytic heme b of the enzyme. Resonance Raman spectra of the fully reduced enzyme are consistent with the presence of two six- coordinate low-spin hemes and one five-coordinate heme b ligated to a histidine. In the low-frequency region of the RR spectrum, a band at 218 cm^{- 1} is assigned to the Fe-N(His) stretching mode of the high-spin heme. Addition of CO induces spectral changes in the high-frequency region of the RR spectra that confirm the binding of CO to the high-spin species. Isotopically labeled CO is used to assign the vibrational modes of the Fe-CO unit: the v(Fe)-co (476 cm^-1) and v(C-O) (1970 cm^-1) as well as the bending mode δ(Fe-C-O) (569 cm^-1). These frequencies show that the catalytic heme is present in an unusual environment, possibly negatively charged, in which CO adopts a geometry quite different from that in cytochrome c oxidase (CcO). The RR study of the oxidized enzyme demonstrates that the high-spin heme b conserves a pentacoordinate structure in the ferric state. To reconcile the EPR data, which indicate the presence of a bridging ligand in the ferric state of the dinuclear center, with the characteristic five-coordinate RR signature of the high-spin heme b in both oxidized and reduced NOR, we propose a mechanism in which the bond between the proximal histidine and the heme iron is broken upon binding of NO, leaving the diiron center bridged after its catalytic turnover.