His92 and His110 selectively affect different heme centers of adrenal cytochrome b561 |
| |
Authors: | Wen Liu Giordano F.Z. da Silva Ah-Lim Tsai Graham Palmer |
| |
Affiliation: | a Department of Internal Medicine, University of Texas Health Science Center at Houston, Houston, Texas, USA b Department of Biochemistry and Cell Biology, Rice University, Houston, Texas, USA c Department of Biochemistry, University of Illinois at Urbana-Champaign, Champaign, Illinois, USA |
| |
Abstract: | Adrenal cytochrome b561 (cyt b561), 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 b561 (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 (bH) peak were seen with mutation of His92; the largest changes in the low-potential (bL) peak lineshape were observed with mutation of His110. In the EPR spectra, mutation of His92 shifted the position of the g = 3.1 signal (bH) but not the g = 3.7 signal (bL). In reductive titrations with ascorbate, mutations in His92 produced the largest increase in the midpoint for the bH transition; mutations in His110 produced the largest decreases in ΔA561 for the bL transition. These results indicate that His92 can be considered part of the bH heme center, and His110 part of the bL heme center, in adrenal cyt b561. |
| |
Keywords: | cyt b561, adrenal cytochrome b561 CG, chromaffin granules CD, circular dichroism EPR, electron paramagnetic resonance HALS, highly axial low-spin EPR signal DM, dodecyl maltoside |
本文献已被 ScienceDirect 等数据库收录! |
|