Chromaffin granule membranes contain at least three heme centers: direct evidence from EPR and absorption spectroscopy

Low-temperature electron paramagnetic resonance (EPR) spectroscopy, circula r dichroism and two-component redox titration have previously provided evid ence for two different ascorbate-reducible heme centers in cytochrome b(561 ) present in chromaffin granule membranes, These species have now been obse rved by room and liquid nitrogen temperature absorption spectroscopy. The v isualization of these heme centers becomes possible as a consequence of uti lizing chromaffin granule membranes prepared by a mild procedure. Additiona lly, a new redox center, not reducible by ascorbate, was discovered by both EPR and absorption spectroscopy. It constitutes about 15% of the heme abso rbance of chromaffin membranes at 561 nm and has EPR characteristics of a w ell-organized highly axial low-spin heme center (thus making it unlikely th at it is a denatured species). This species is either an alternative form o f one of the hemes of cylochrome: b(561) that has a very low redox potentia l or a b-type cytochrome distinct from b(561) (C) 2001 Federation of Europe an Biochemical Societies. Published by Elsevier Science B.V. All rights res erved.