Postnikova G B, Shekhovtsova E A
Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia.
Biochemistry (Mosc). 2018 Feb;83(2):168-183. doi: 10.1134/S0006297918020098.
In this review, we shortly summarize the data of our studies (and also corresponding studies of other authors) on the new mechanism of myoglobin (Mb) deoxygenation in a cell, according to which Mb acts as an oxygen transporter, and its affinity for the ligand, like in other transporting proteins, is regulated by the interaction with the target, in our case, mitochondria (Mch). We firstly found that contrary to previously formulated and commonly accepted concepts, oxymyoglobin (MbO) deoxygenation occurs only via interaction of the protein with respiring mitochondria (low p values are necessary but not sufficient for this process to proceed). Detailed studies of the mechanism of Mb-Mch interaction by various physicochemical methods using natural and artificial bilayer phospholipid membranes showed that: (i) the rate of MbO deoxygenation in the presence of respiring Mch fully coincides with the rate of O2 uptake by mitochondria from a solution irrespectively of their state (native coupled, freshly frozen, or FCCP-uncoupled), i.e. it is determined by the respiratory activity of Mch; (ii) Mb nonspecifically binds to membrane phospholipids of the outer mitochondrial membrane, while any Mb-specific protein or phospholipid sites on it are lacking; (iii) oxygen uptake by Mch from a solution and the uptake of Mb-bound oxygen are two different processes, as their rates are differently affected by proteins (e.g. lysozyme) that compete with MbO for binding to the mitochondrial membrane; (iv) electrostatic forces significantly contribute to the Mb-membrane interactions; the dependence of these interactions on ionic strength is provided by the local electrostatic interactions between anionic groups of phospholipids (the heads) and invariant Lys and Arg residues near the Mb heme pocket; (v) interactions of Mb with phospholipid membranes promote conformational changes in the protein, primarily in its heme pocket, without significant alterations in the protein secondary and tertiary structures; and (vi) Mb-membrane interactions lead to decrease in the affinity of myoglobin for O2, which could be monitored by the increase in the MbO autooxidation rate under aerobic conditions and under anaerobic ones, by the shift in the MbO/Mb(2) equilibrium towards the ligand-free protein. The decrease in the affinity of Mb for the ligand should facilitate O2 dissociation from MbO at physiological p values in cells.
在本综述中，我们简要总结了我们的研究（以及其他作者的相应研究）中关于细胞内肌红蛋白（Mb）脱氧新机制的数据，据此Mb作为氧转运蛋白，并且其对配体的亲和力，如同其他转运蛋白一样，通过与靶标（在我们的研究中为线粒体（Mch））的相互作用来调节。我们首先发现，与先前提出并被普遍接受的概念相反，氧合肌红蛋白（MbO）的脱氧仅通过蛋白质与进行呼吸作用的线粒体的相互作用发生（低p值对于该过程的进行是必要的，但并不充分）。使用天然和人工双层磷脂膜通过各种物理化学方法对Mb - Mch相互作用机制进行的详细研究表明：（i）在存在进行呼吸作用的Mch的情况下，MbO的脱氧速率与线粒体从溶液中摄取O2的速率完全一致，而与它们的状态（天然偶联、新鲜冷冻或FCCP - 解偶联）无关，即它由Mch的呼吸活性决定；（ii）Mb非特异性地结合到线粒体外膜的膜磷脂上，而其上不存在任何Mb特异性的蛋白质或磷脂位点；（iii）Mch从溶液中摄取氧气和摄取与Mb结合的氧气是两个不同的过程，因为它们的速率受到与MbO竞争结合线粒体膜的蛋白质（例如溶菌酶）的不同影响；（iv）静电力对Mb - 膜相互作用有显著贡献；这些相互作用对离子强度的依赖性由磷脂（头部）的阴离子基团与Mb血红素口袋附近不变的赖氨酸和精氨酸残基之间的局部静电相互作用提供；（v）Mb与磷脂膜的相互作用促进蛋白质构象变化，主要是在其血红素口袋中，而蛋白质的二级和三级结构没有明显改变；并且（vi）Mb - 膜相互作用导致肌红蛋白对O2的亲和力降低，这可以通过在有氧条件和无氧条件下MbO自氧化速率的增加、MbO/Mb(2)平衡向无配体蛋白的移动来监测。Mb对配体亲和力的降低应有助于在细胞生理p值下O2从MbO上解离。
