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Axial ligation and stoichiometry of heme centers in adrenal cytochrome b561.

ABSTRACT: Cytochrome (cyt) b561 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 b561 using electron paramagnetic resonance (EPR) and optical spectroscopy on the wild-type and mutant forms of the cytochrome expressed in insect cells. The gz = 3.7 (low-potential heme) and gz = 3.1 (high-potential heme) signals were found to represent the only two authentic hemes of cyt b561; models that propose smaller or greater amounts of heme can be ruled out. We identified the axial ligands to hemes in cyt b561 by mutating four conserved histidines (His54 and His122 at the matrix-side heme center and His88 and His161 at the cytoplasmic-side 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 b561 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 gz = 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.

SUBMITTER: Kamensky Y

PROVIDER: S-EPMC2551744 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature

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Axial ligation and stoichiometry of heme centers in adrenal cytochrome b561.

Kamensky Yury Y Liu Wen W Tsai Ah-Lim AL Kulmacz Richard J RJ Palmer Graham G

Biochemistry 20070630 29

Cytochrome (cyt) b561 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 b561 using electron paramagnetic resonance (EPR) and optical spectroscopy on the wild-type and mutant forms of the cytochrome expressed in insect cells. The gz = 3.7 (low-potential heme) and gz = 3.1 (high-potential heme) signals were found to represent the on ...[more]

PMID: 17602662

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Project description:Several residues in the third extramembrane segment (EM3) of adrenal cytochrome b(561) have been proposed to be involved in this cytochrome's interaction with ascorbate, but there has been no systematic evaluation of residues in the segment. We used alanine scanning mutagenesis to assess the functional and structural roles of the EM3 residues and several adjacent residues (residues 70-85) in the bovine cytochrome. Each alanine mutant was expressed in a bacterial system, solubilized with detergent, and affinity-purified. The recombinant proteins contained approximately two hemes per monomer and, except for R74A, retained basic functionality (? 94% reduced by 20 mM ascorbate). Equilibrium spectrophotometric titrations with ascorbate were used to analyze the ?-band line shape and amplitude during reduction of the high- and low-potential heme centers (b(H) and b(L), respectively) and the midpoint ascorbate concentrations for the b(H) and b(L) transitions (C(H) and C(L), respectively). Y73A and K85A markedly narrowed the b(H) ?-band peak; other mutants had weaker effects or no effect on b(H) or b(L) spectra. Relative changes in C(H) for the mutants were larger than changes in C(L), with 1.5-2.9-fold increases in C(H) for L70A, L71A, Y73A, R74A, N78A, and K85A. The amounts of functional b(H) and b(L) centers in additional Arg74 mutants, assessed by ascorbate titration and EPR spectroscopy, declined in concert in the following order: wild type > R74K > R74Q > R74T and R74Y > R74E. The results of this first comprehensive experimental test of the proposed roles of EM3 residues have identified residues with a direct or indirect impact on ascorbate interactions, on the environment of the b(H) heme center, and on formation of the native b(H)-b(L) unit. Surprisingly, no individual EM3 residue was by itself indispensable for the interaction with ascorbate, and the role of the segment appears to be more subtle than previously thought. These results also support our topological model of the adrenal cytochrome, which positions b(H) near the cytoplasmic side of the membrane.

Dataset Information

Axial ligation and stoichiometry of heme centers in adrenal cytochrome b561.

Publications

Axial ligation and stoichiometry of heme centers in adrenal cytochrome b561.

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