Postnikova G B, Shliapnikova E A, Atanasov B P
Mol Biol (Mosk). 1982 Jan-Feb;16(1):104-16.
The influence of chemical modification of His residues in Mb on the rate of redox reaction in system MbO2--Cyt c has been studied at different ionic strengths and pH medium. The products of alkylation of all available His by bromacetate and iodacetamide, CM-Mb and CA-Mb, respectively, and myoglobin, modified by spin label 2,2', 6,6'-tetramethyl-4-bromoacetoxypiperidine-1-oxyl (SL) at His residue A10--Sl (His-A10)--Mb have been studied. It has been shown, that the character of the ionic strength dependence of reaction SL(His-A10)--MbO2 with Cyt c at pH 6.0 ann 7.0 is basically analogues to that, observed for intact protein. It means that only His-GH1 of two His residues, His-A10 and His-GH1, situated in the region of "active contact" of Mg with Cyt c molecule, participates in the interactions, essential for electron transfer. The interaction of the charge of this His with the negatively charged group of Cyt c is necessary, probably for the proper arrangement of other interactions in the active complex, because the deprotonation of His-GHl in the studied pH interval decreases the rate of the process by more than one order of magnitude. The rate of oxidation of MC-MbO2 and CA-MbO2 by ferricytochrome c, in contrast to intact protein, shows a weak dependence on the ionic strength and does not depend on the pH medium, throughout the range of ionic strengths from 0.005 to 1.0. The cause of the radical change in the ionic strength dependence is, probably, nearly entire disturbance of electrostatic interactions in the active complex due to chemical modification of His residues in the site of "active contact", and first of all, the His-CHl residue. The fact, that during alkylation of all available His in Mb the electron transfer persists in the system, points to that in the process of electron transfer to cytochrome c, uncharged group, most probably "inner" His-B5, participates. Based on the data on spatial structure and the obtained results, the positions of the charged groups in the site of "active contact" of Mb with Cyt c molecule are presented.
在不同离子强度和pH介质条件下，研究了肌红蛋白（Mb）中组氨酸（His）残基的化学修饰对体系MbO₂⁻-细胞色素c（Cyt c）中氧化还原反应速率的影响。分别研究了用溴乙酸盐和碘乙酰胺对所有可及His进行烷基化的产物，即羧甲基化肌红蛋白（CM-Mb）和羧酰胺甲基化肌红蛋白（CA-Mb），以及在His残基A10处用自旋标记物2,2',6,6'-四甲基-4-溴乙酰氧基哌啶-1-氧基（SL）修饰的肌红蛋白，即SL(His-A10)-Mb。结果表明，在pH 6.0和7.0条件下，反应SL(His-A10)-MbO₂与Cyt c的离子强度依赖性特征与完整蛋白质的基本类似。这意味着位于Mb与Cyt c分子“活性接触”区域的两个His残基，即His-A10和His-GH1中，只有His-GH1参与了对电子转移至关重要的相互作用。该His的电荷与Cyt c带负电基团的相互作用可能是活性复合物中其他相互作用正确排列所必需的，因为在所研究的pH范围内His-GH1的去质子化使反应速率降低了一个多数量级以上。与完整蛋白质相比，高铁细胞色素c对MC-MbO₂和CA-MbO₂的氧化速率在离子强度从0.005到1.0的整个范围内对离子强度的依赖性较弱，且不依赖于pH介质。离子强度依赖性发生根本变化的原因可能是由于“活性接触”位点处His残基的化学修饰，尤其是His-CH1残基，导致活性复合物中的静电相互作用几乎完全受到干扰。在Mb中所有可及His烷基化过程中电子转移在体系中持续存在这一事实表明，在向细胞色素c的电子转移过程中，不带电基团，很可能是“内部”的His-B5参与其中。基于空间结构数据和所得结果，给出了Mb与Cyt c分子“活性接触”位点处带电基团的位置。
