DIFFERENTIAL CHARACTERIZATION OF NEUTROPHIL CYTOCHROME P-30 AND CYTOCHROME B-558 BY LOW-TEMPERATURE ABSORPTION AND RESONANCE RAMAN SPECTROSCOPIES

Cytochrome p30, a novel hemoprotein isolated from rabbit peritoneal ne utrophils [Escriou, V., Laporte, F. Garin, J., Brandolin, G. & Vignais , P. V. (1994) J. Biol. Chem. 269, 14007-14014] has been characterized by low-temperature (77 K) absorption and resonance Raman spectroscopi es. The spectral data have been compared with those obtained with neut rophil cytochrome b-558. At room temperature, the absorption differenc e spectra (reduced minus oxidized) of cytochrome p30 and cytochrome b- 558 could not been distinguished from each other. However, at 77 K, si gnificant differences were observed. In particular, the a band of cyto chrome p30 was split whereas that of cytochrome 6-558 was symmetrical, but particularly broad. The resonance Raman spectra of cytochrome p30 provided evidence for the presence of two hemes both in the ferric an d ferrous states. One of them was a six-coordinated low-spin heme eith er oxidized or reduced whereas the other one was a high-spin heme, fiv e-coordinated in the reduced state and six-coordinated in the oxidized state. It is probable that two histidine residues constitute the axia l 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 p3 0. The component typical of the high-spin heme of cytochrome p30 was h owever absent in the spectra of oxidized and reduced cytochrome b-558.