Human neutrophil cytochrome b contains multiple hemes. Evidence for heme associated with both subunits.
Human phagocyte cytochrome b is the terminal component of the microbicidal superoxide generating system. Although the primary structure of this protein has been determined, little is known about the placement of the heme prosthetic groups in this heterodimeric integral membrane protein. Analysis of the cytochrome using lithium dodecyl sulfate-polyacrylamide gel electrophoresis at 0 degree C followed by tetramethylbenzidine heme staining demonstrated the presence of heme in both the 91- and 22-kDa subunits identified by Western blot analysis using peptide specific antisera. Exposure of cytochrome b (purified or in isolated neutrophil plasma membranes) to Staphylococcal protease V8 or trypsin did not affect absorbance spectra. However, such treatment resulted in degradation of both subunits to smaller fragments, including characteristic immunoreactive 20-kDa fragments of both the large and small subunits of the cytochrome that retained one or both of the hemes. The spectral stability to proteolysis and size of the proteolytic heme-containing fragments generated explains previous reports which suggested that the heme resided in the small subunit. Our current results indicate that human neutrophil cytochrome b is a bi-heme or possibly tri-heme molecule with at least one heme residing in the large subunit and one shared between both subunits and that the heme-containing regions of the cytochrome probably lie within the membrane lipid bilayer. Such a multi-heme structure would be consistent with an electron transfer function for this cytochrome by providing an efficient mechanism for transferring electrons across the plasma membrane to the extracellular surface where oxygen could be reduced to create superoxide.
Quinn
M T
MT
Department of Chemistry and Biochemistry, Montana State University, Bozeman 59717.
Mullen
M L
ML
Jesaitis
A J
AJ
D000595:Amino Acid Sequence; D001560:Benzidines; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D002863:Chromogenic Compounds; D003573:Cytochrome b Group / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology*
D001560:Benzidines; D002863:Chromogenic Compounds; D003573:Cytochrome b Group; C021758:3,3',5,5'-tetramethylbenzidine; D006418:Heme