Differential characterization of neutrophil cytochrome p30 and cytochrome b-558 by low-temperature absorption and resonance Raman spectroscopies

Abstract

Cytochrome p30, a novel hemoprotein isolated from rabbit peritoneal neutrophils [Escriou, V., Laporte, E, Garin, J., Brandolin, G. and Vianais, P.V. (1994) J. Biol. Chem. 269, 14007-14014] has been characterized by low-temperature (77 K) absorption and resonance Raman spectroscopies. The spectral data have been compared with those obtained with neutrophil cytochrome b-558. At room temperature, the absorption difference spectra (reduced minus oxidized) of cytochrome p30 and cytochrome b-558 could not been distinguished from each other. However, at 77 K, significant differences were observed. In particular, the α band of cytochrome p30 was split whereas that of cytochrome b-558 was symmetrical, but particularly broad. The resonance Raman spectra of cytochrome p30 provided evidence for the presence of two hemes both in the ferric and ferrous states. One of them was a six-coordinated low-spin heme either oxidized or reduced whereas the other one was a high-spin heme, five-coordinated in the reduced state and six-coordinated in the oxidized state. It is probable that two histidine residues constitute the axial ligands of the six-coordinated low-spin heme of cytochrome p30. The resonance Raman spectra of cytochrome b-558 allowed the detection of a six-coordinated low-spin heme, similar to that found in cytochrome p30. The component typical of the high-spin heme of cytochrome p30 was however absent in the spectra of oxidized and reduced cytochrome b-558.