His92 and His110 selectively affect different heme centers of adrenal cytochrome b561

Abstract

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).

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

“…As expected from previous results (7, 16) the heme content of wildtype cyt b 561 was almost exactly two per monomer (Supplementary Material, Table S2), showing that most modifications of EM3 sidechains did not prevent incorporation of a full heme complement into the protein. The basic functionality of each recombinant protein was assessed by comparison of the intensity of alpha band absorbance from cytochrome reduction upon addition of 20 mM ascorbate to that upon complete reduction by dithionite (Supplementary Material, Table S2; full spectra of wildtype cytochrome and selected mutants are in Fig.…”

“…Ascorbate titrations were performed as previously published with minor changes (7). Pre-oxidized cyt b 561 samples (5–11 μM) in 100 mM potassium phosphate buffer (pH 7.2) containing 18% glycerol and 0.08% (w/v) n-dodecyl-β-D-maltoside were placed in a 1-cm quartz cuvette.…”

“…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.…”

Cytochromesb561: Ascorbate-Mediated Trans-Membrane Electron Transport

“…Mutations in other conserved and partially conserved residues of mouse CYB561s had variable effects on TMET activity and ASC reduction (34,41,45,54). Consistent with the putative location of the electron-accepting and -donating sides, mutations in loop regions on the intra-vesicular side severely decreased LCytb/CYB561A3 TM Fe 3 + -reductase activity, but mutations on the cytoplasmic side had comparatively less effect (54).…”

“…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).…”