Resonance Raman detection of a ferrous five-coordinate nitrosylheme b3 complex in cytochrome cbb3 oxidase from Pseudomonas stutzeri

Understanding the chemical nature of the nitric oxide (NO) moiety of nitrosylheme copper oxidases is crucial for elucidation of the NO activation process. In the present work, direct resonance Raman spectroscopic observation of both the Fe²⁺-NO and the N-O stretching modes unambiguously establishes the vibrational characteristics of the NO-bound heme moiety in cytochrome cbb₃ from Pseudomonas stutzeri. Addition of NO to fully reduced enzyme causes the rupture of the proximal His-heme b3 bond resulting in the formation of a five-coordinate heme b₃²⁺-NO species with ν(Fe-NO) and ν(NO) at 524 and 1679 cm^-1, respectively. The frequencies of the nitrosyl species we detect are very similar to those obtained in other model- and protein heme-NO complexes. To account for this observation, we propose a model describing the oxidation and ligand-binding states in fully reduced cytochrome cbb₃ upon addition of NO.