Fourier Transform Infrared and Resonance Raman Studies of the Interaction of Azide with Cytochrome c Oxidase from Paracoccus denitrificans

Abstract

The interaction of azide with oxidized cytochrome c oxidase from Paracoccus denitrificans has been studied by resonance Raman, Fourier transform infrared, and UV−vis spectroscopy. Azide binds in two phases: a high-affinity phase (Kd = 4.1 μM) in which it is bound to a nonmetal site near the binuclear center and a low-affinity phase (Kd = 11.4 mM) in which it is bound as a bridge to the binuclear center. The resonance Raman spectra of the low-affinity phase display one isotope-dependent vibrational mode at 417 cm-1. The FTIR spectra display two isotope-dependent bands at 2038 and 2056 cm-1. We assign the band at 417 cm-1 to ν(Fe−N3−CuB) and the bands at 2038 and 2056 cm-1 to νas(N3). We observe similar FTIR spectra for the azide complex of bovine heart oxidase and conclude that the binuclear center in this oxidase behaves in a manner analogous to the P. denitrificans enzyme. In contrast to mammalian cytochrome c oxidase (Li, W.; Palmer, G. Biochemistry 1993, 322, 1833−1843), the low-affinity phase observed in the interaction of azide with the P. denitrificans enzyme is not associated with binding of azide to heme a. The observation of two FTIR νas(N3) modes suggests that the azide ion binds to two different enzyme conformations, both forming bridging complexes with the binuclear center. Comparison of the UV−vis, resonance Raman, and FTIR data of the azide-bound cytochrome c aa3 from P. denitrificans and those of azide-bound quinol cytochrome bo3 suggest significant alterations in the interaction of azide with the oxidized forms of these bacterial oxidases resulting from specific structural differences within their respective heme a3−CuB and heme o3−CuB binuclear pockets.