Abstract
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.
Highlights
The resonance Raman spectra of neutrophil cyto- physiological heme-bearing protein
A wide variety of experimental evidence [7] suggests thatitfunctionsasa specific granules, as were spectra from unstimulated component of the NADPHoxidase, a unique respiratory chain and phorbol myristate acetate-stimulated cells, indi- in the neutrophil plasma membranwehich, when triggeredby cating that thheemes are structurallyidentical invar- stimulant binding to the external surface, initiates one-elecious subcellular environments and cellular physiologi-tron reduction of O2 [8]
We present structural information germane electrolyzing 1.5mM anthraquinone-2,6-disulfonateuntil thesolution to these issues derived from resonance Raman spectroscopy on cyt bnnHb, oth within the plasma membrane ansdecondary granule fractions and as asolubilized fraction
Summary
Isolation of Subcellular Fractions-Specific granules isolated as of 0.1 mM cytochrome c in 0.1 M phosphate, pH 7.0, 0.1 M NaCl with previously described [33] were used as a source for cyt b85R.The 15 p~ dichlorophenolindolphenol as mediator-titrant gave alinear same solubilization technique describedabovefor membranes was Nernst plot obtained from optical difference spectra of the a-band employed. Were added as 7 p1 of 2 mM aqueous solutions each to final mediatortitrant concentrations of about 15PM,Nernst plots were constructed from optical difference spectra obtained by cyclic reductive-oxidative titrations with S,O:- and Fe(CN)g- as redox agents; data were corrected for dilution by the titrant,which constituted about 10% of the total volume/half-cycle. Granules, and whole cells were pelleted by spinning capillary tubes containing the suspensions in a hematocrit centrifuge prior to sealing the capillaries.
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