Cytochrome (cyt) b 561 transports electrons across the membrane of chromaffin granules (CG) present in the adrenal medulla, supporting the biosynthesis of norepinephrine in the CG matrix. We have conducted a detailed characterization of cyt b 561 using electron paramagnetic resonance (EPR) and optical spectroscopy on the wild type and mutant forms of the cytochrome expressed in insect cells. The g z = 3.7 (low-potential heme) and g z = 3.1 (high-potential heme) signals were found to represent the only two authentic hemes of cyt b 561 ; models that propose smaller or greater amounts of heme can be ruled out. We identified the axial ligands to hemes in cyt b 561 by mutating four conserved histidines (His54 and His122 at the matrix-side heme center and His88 and His161 at the cytoplasmicside heme center), thus confirming earlier structural models. Single mutations of any of these histidines produced a constellation of spectroscopic changes that involve not one but both heme centers. We hypothesize that the two hemes and their axial ligands in cyt b 561 are integral parts of a structural unit that we term the "kernel". Histidine to glutamine substitutions in the cytoplasmic-side heme center, but not in the matrix-side heme center, led to the retention of a small fraction of the low-potential heme with g z = 3.7. We provisionally assign the low-potential heme to the matrix side of the membrane; this arrangement suggests that the membrane potential modulates electron transport across the CG membrane.Most, if not all, eukaryotic organisms carry a gene for at least one member of the newly discovered protein family of cytochrome b 561 (cyt b 561 ) 1 (1-4). The oldest known member of the family, adrenal-type cyt b 561 , is essentially an ascorbate-regeneration machine (5) that plays a key role in supplying electrons for the biosynthesis of norepinephrine by dopamine-β-hydroxylase (6) and of several important peptide hormones by peptidylglycine β-monooxygenase (6). Several other cyt b 561 family members are either suggested to participate in ascorbate metabolism or to depend on ascorbate for ferric-reductase activity. Among them † This work was supported by American Heart Association (Texas Affiliate) Grant 0455107Y (G.P.), National Institutes of Health GM44911 (A-L.T.). * Address correspondence to these authors: Y. Kamensky, Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251, . E-mail: yuryk@bioc.rice.edu G. Palmer, Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251, USA. Telephone: 713-348-4860. FAX: 713-348-5154. E-mail: graham@bioc.rice.edu. 3 Our earlier publication (Kamensky, Y.A. and Palmer, G. (2001) Chromaffin granule membranes contain at least three heme centers, FEBS Lett. 491,) considered a third heme as either a cyt b 561 component or another heme-containing protein present in the CG membrane. That this third heme is not a part of cyt b 561 itself is now confirmed, as neither purified cyt b 561 from CG nor those expressed in insect, yeast or bac...

Adrenal cytochrome b(561) (cyt b(561)), a transmembrane protein that shuttles reducing equivalents derived from ascorbate, has two heme centers with distinct spectroscopic signals and reactivity towards ascorbate. The His54/His122 and His88/His161 pairs furnish axial ligands for the hemes, but additional amino acid residues contributing to the heme centers have not been identified. A computational model of human cyt b(561) (Bashtovyy, D., Berczi, A., Asard, H., and Pali, T. (2003) Protoplasma 221, 31-40) predicts that His92 is near the His88/His161 heme and that His110 abuts the His54/His122 heme. We tested these predictions by analyzing the effects of mutations at His92 or His110 on the spectroscopic and functional properties. Wild type cytochrome and mutants with substitutions in other histidine residues or in Asn78 were used for comparison. The largest lineshape changes in the optical absorbance spectrum of the high-potential (b(H)) peak were seen with mutation of His92; the largest changes in the low-potential (b(L)) peak lineshape were observed with mutation of His110. In the EPR spectra, mutation of His92 shifted the position of the g=3.1 signal (b(H)) but not the g=3.7 signal (b(L)). In reductive titrations with ascorbate, mutations in His92 produced the largest increase in the midpoint for the b(H) transition; mutations in His110 produced the largest decreases in DeltaA(561) for the b(L) transition. These results indicate that His92 can be considered part of the b(H) heme center, and His110 part of the b(L) heme center, in adrenal cyt b(561).

No abstract

No abstract