Fourier Transform Infrared Evidence for a Ferric Six-Coordinate Nitrosylheme b3 Complex of Cytochrome cbb3 Oxidase from Pseudomonas Stutzeri at Ambient Temperature

Abstract

We report the first vibrational study of NO bound to an oxidized heme−copper oxidase. Cytochrome cbb3 oxidase from P. stutzeri reduces both O2 and NO to H2O and N2O, respectively. The ferric nitrosyl complex of cbb3 exhibits ν(N−O) at 1903 cm-1. This frequency is very similar to ν(NO) of nitric oxide reductase, the acidic form of Met Mb−NO, but 18 cm-1 lower than that of neutral Met Mb−NO. By monitoring the NO intensity, we estimate that NO dissociates from the heme b3 pocket, without binding to CuB, with k = 1.8 × 10-3 s-1. Therefore, NO binding occurs at the heme site and not at CuB, generating a nitrosonium CuB1+−NO+ species as proposed recently (Torres, J.; Cooper, C. E.; Wilson, M. T. J. Biol. Chem. 1998, 273, 8756−8766). The coordination of NO to cbb3 oxidase and to nitric oxide reductase and Mb is compared and discussed.