Axial Ligation and Stoichiometry of Heme Centers in Adrenal Cytochromeb₅₆₁

Abstract

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...

Cytochromesb561: Ascorbate-Mediated Trans-Membrane Electron Transport

“…Spectrum analysis of mouse and bovine CGCytb/CYB561A1 and of TSCytb/CYB561D2 revealed the presence of two distinct, split a-bands in the spectrum of ASC-reduced proteins, which is consistent with the presence of two heme pockets (9,12,28). In Arabidopsis TCytb/CYB561B1 and mouse TSCytb/CYB561D2, singular value decomposition analysis identified two distinct b-type heme spectra that could be assigned to the two CYB561 hemes (12,16).…”

“…Western blot analysis and spectroscopy demonstrated that mutation of His residues coordinating the LP-heme (intra-vesicular-side) of Arabidopsis TCytb/ CYB561B1, and mouse CGCytb/CYB561A1, resulted in nearly undetectable protein levels. In contrast, mutations in the HPheme-coordinating residues hardly affected CYB561 expression, but resulted in altered ASC-reduction kinetics and reduced heme content (11,28,34). Somewhat contradicting results were obtained with His mutations in human DCytb/CYB561A2, and need further investigation (37,45).…”

“…Direct molecular-binding interactions between ASC and CYB561 have not been measured, but steady-state and fast kinetic measurements on plant and animal CYB561s suggest the existence of two distinct ASC-'affinity' sites, with approximate values of 0.01 mM and 1 mM, for CGCytb/ CYB561A1 (7,11,28). These parameters are not real binding constants but rather characterize the interaction between ASC .…”

Functional and Structural Roles of Residues in the Third Extramembrane Segment of Adrenal Cytochrome b561

“…The positions of the b H and b L centers in the chromaffin granule membrane have been controversial, with one model assigning b H to the His88/His161 heme (as depicted in Fig. 1) (6, 7), and an earlier model assigning b H to the His54/His122 heme (8, 9). A key issue from a mechanistic perspective is whether it is b H or b L that is exposed to the cytoplasm and its pool of ascorbate.…”

Auxin-Responsive Genes AIR12 Code for a New Family of Plasma Membrane b-Type Cytochromes Specific to Flowering Plants

“…Cytochromes b561 are high-potential, transmembrane redox proteins of about 25 kD made of six membrane-spanning a-helices, which bind two hemes b. One heme is predicted to be close to an ascorbate binding site facing the cytosol, whereas the second heme faces the opposite side of the membrane and can be oxidized by either MDA or ferrichelates (Tsubaki et al, 1997;McKie et al, 2001;Bérczi et al, 2005;Kamensky et al, 2007). Plants contain several orthologous genes to animal cytochrome b561 Bashtovyy et al, 2003).…”