Effect of heme orientation on the reduction potential of cytochrome b5.
Effect of heme orientation on the reduction potential of cytochrome b5.
复制标题
血红素取向对细胞色素b5还原电位的影响。
DOI：
10.1021/ja00226a045
复制
发表时间：
1988
影响因子：
15
通讯作者：
D. H. Buttlaire
中科院分区：
文献类型：
--
作者：
F. Walker;David Emrick;Jenny E. Rivera;B. Hanquet;D. H. Buttlaire
Bovine erythrocyte cytochrome b,, a soluble protein, and bovine microsomal cytochrome b,, solubilized by trypsin cleavage, were isolated and purified, and their proton NMR spectra were compared. These two proteins had identical chemical shifts of the heme resonances and the same ratio of major to minor heme orientation. Thus, spectroelectrochemical titrations were carried out on the trypsin-cleaved microsomal protein. In its equilibrium ratio of 9:l major to minor heme orientation, cytochrome bS gave a reduction potential of -1.9 f 1.6 mV vs SHE at p = 0.13 M, pH 7.0, and T = 24 OC, utilizing three electrochemical mediators, 0.2 mM Ru(NH&CI3, 0.2 mM K3Fe(CN)6 and 1 mM methyl viologen. Spectroelectrochemical titrations of the apoprotein freshly reconstituted with hemin, in which the majorminor ratio was close to l:l, yielded a reduction potential of -10.0 f 1.7 mV vs SHE under the conditions listed above. The potentials of two samples of reconstituted cytochrome bS that had been allowed to equilibrate for 3 days were both -1.8 mV, indicating a return to the equilibrium ratio of major to minor forms. NMR quantitation of the time course of heme rotation in H20 under the electrochemical conditions has led to an estimate of major:minor ratio of 60:40 at the midpoint of the titration and a half-life for heme reorientation of 12 f 1 h. These data lead to calculated reduction potentials of +0.8 and -26.2 mV, respectively, for pure major and minor heme orientations. Although this difference is probably not large enough to be physiologically significant, the fact that the difference exists suggests that a detailed correlation between structure and reduction potential is very important for understanding the reduction potentials of heme proteins in general. Cytochrome bS is a heme protein that exists in soluble form in erythrocytes and in membrane-bound form in liver microsomes. Erythrocyte cytochrome bS has been shown to mediate the re- duction of methemoglobin by NADH-cytochrome b5 reductase in normal red cell~.I-~ The presence of cytochrome b, in erythrocytes stimulates methemoglobin reduction by NADH- cytochrome bs reductase by as much as 77-fold.' The protein is thus important in reducing the ca. 3% of hemoglobin that is oxidized to the met form each day in normal humans? Low levels of erythrocyte cytochrome b, may be associated with some forms of methemoglobinemia.' Microsomal cytochrome bS is a membrane-bound amphiphatic redox protein that functions as a component of a microsomal electron-transfer chain in endoplasmic reticulum membranes. It has also been shown to be bound to outer mitochondrial mem- brane~~ and in plasma membranes from intestinal microvilli and erythrocytes6 as well as in the mitochondrial intramembrane space.' There is evidence that it participates in the microsomal stearyl-CoA desaturation reaction.' It is also known to interact with cytochrome P450 as an alternate electron donor and to act as a respiratory carrier during hepatic microsomal mixed-function oxidation reactions.' It is a two-domain protein, with a hydro-
