Resonance Raman and electron paramagnetic resonance structural investigations of neutrophil cytochrome b558.
The resonance Raman spectra of neutrophil cytochrome b558 obtained upon Soret excitation indicate that the heme is low spin six-coordinate in both ferric and ferrous oxidation states; comparison with the spectra of bis-imidazole hemin suggests imidazole or imidazolate axial ligation. Minor bands attributable to vibrational motions of ring-conjugated vinyl substituents were also observed, consistent with a heme assignment of protoporphyrin IX. The spectra of deoxycholate-solubilized cytochrome b558 were indistinguishable from neutrophil plasma membranes or specific granules, as were spectra from unstimulated and phorbol myristate acetate-stimulated cells, indicating that the hemes are structurally identical in various subcellular environments and cellular physiological states. However, structural complexity was suggested by biphasic ferric-ferrous photoreduction under 413-nm illumination and the absence of an EPR spectrum for the ferric heme under conditions where simple bis-imidazole heme-containing cytochromes are expected to give detectable signals. Midpoint reduction potentials and resonance Raman spectra of the soluble cytochrome b558 from an individual with cytochrome b558 positive (type IA.2) chronic granulomatous disease were nearly identical to normal oxidase, with the exception that the deficient oxidase did not undergo heme photoreduction. Possible structural models are discussed in relation to other physical properties (ligand binding, thermodynamic potentials) exhibited by the cytochrome.
Hurst
J K
JK
Department of Chemical and Biological Sciences, Oregon Graduate Institute of Science and Technology, Beaverton 97006-1999.
Loehr
T M
TM
Curnutte
J T
JT
Rosen
H
H
D002462:Cell Membrane / Q000201:enzymology; D003573:Cytochrome b Group / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000201:enzymology; D015394:Molecular Structure; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010084:Oxidation-Reduction; D011166:Porphyrins / Q000737:chemistry; D013059:Spectrum Analysis, Raman
D003573:Cytochrome b Group; D011166:Porphyrins; D006418:Heme; C036888:cytochrome b558; D019255:NADPH Oxidases