Control of formation and dissociation of the high-affinity complex between cytochrome c and cytochrome c peroxidase by ionic strength and the low-affinity binding site.
Control of formation and dissociation of the high-affinity complex between cytochrome c and cytochrome c peroxidase by ionic strength and the low-affinity binding site.
复制标题
通过离子强度和低亲和力结合位点控制细胞色素 c 和细胞色素 c 过氧化物酶之间高亲和力复合物的形成和解离。
DOI：
10.1021/bi961487k
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Millett,F
中科院分区：
文献类型：
--
作者：
Mei,H;Wang,K;McKee,S;Wang,X;Waldner,JL;Pielak,GJ;Durham,B;Millett,F
用一种新的Ru光还原技术测定了酵母异-1-细胞色素(YCC)与细胞色素过氧化物酶I(CMPI)在较宽的离子强度范围内高亲和力络合物的形成和解离速率常数。这些研究利用了Ru-39-CC，它含有连接在YCC H39C，C102T变种半胱氨酸残基上的三联吡啶Ru，并与CMPI具有与天然yCcc相同的反应活性。kdf是在氧化的yCcIII：CMPI络合物存在下通过光还原少量Ru-39-CC测量的，该络合物必须在Ru-39-CcII与CMPI结合并还原自由基阳离子之前解离。在低离子强度下，1：1高亲和力络合物的kd值很小，但通过将yCC与第二个低亲和力位点结合，kd值显著增加。然而，在CMPI中，低亲和力的yCC结合部位在直接电子转移到自由基阳离子或氧铁血红素方面不活跃，在离子强度大于70 mM时太弱，不能在动力学中发挥作用。在150 mm离子强度下，kd值增加到4000 S-1，而在低离子强度下，kd值从3×109m-1s-1下降到150 mm离子强度下的1.3×109m-1s-1。这些研究表明，酶转换的限速步骤是在150 mM离子强度以下的产物解离和在较高离子强度时向氧铁血红素的复合体内电子转移。YCC和CCP之间的相互作用是在生理离子强度下优化的，以提供最大可能的络合物形成速率常数Kf，而不允许产品解离受到速率限制。表面突变对动力学的影响提供了证据，表明溶液中反应所用的高亲和力结合部位与YCC：CCP晶体结构中鉴定的相似。
A new ruthenium photoreduction technique was used to measure the formation and dissociation rate constantskfandkdof the high-affinity complex between yeast iso-1-cytochromec(yCc) and cytochromecperoxidase compound I (CMPI) over a wide range of ionic strength. These studies utilized Ru-39-Cc, which contains trisbipyridylruthenium attached to the cysteine residue in the H39C,C102T variant of yCc, and has the same reactivity with CMPI as native yCc.kdandkfwere measured by photoreducing a small concentration of Ru-39-Ccin the presence of the oxidized yCcIII:CMPI complex, which must dissociate before Ru-39-CcIIcan bind to CMPI and reduce the radical cation. The value ofkdfor the 1:1 high-affinity complex is very small at low ionic strength, <5 s-1but is increased significantly by binding yCcto a second low-affinity site. However, the low-affinity yCcbinding site is not active in direct electron transfer to either the radical cation or the oxyferryl heme in CMPI, and is too weak to play a role in the kinetics at ionic strengths above 70 mM. The value ofkdincreases to 4000 s-1at 150 mM ionic strength, whilekfdecreases from >3 × 109M-1s-1at low ionic strength to 1.3 × 109M-1s-1at 150 mM ionic strength. These studies indicate that the rate-limiting step in enzyme turnover is product dissociation below 150 mM ionic strength and intracomplex electron transfer to the oxyferryl heme at higher ionic strength. The interaction between yCcand CcP is optimized at physiological ionic strength to provide the largest possible complex formation rate constantkfwithout allowing product dissociation to be rate-limiting. The effects of surface mutations on the kinetics provided evidence that the high-affinity binding site used for the reaction in solution is similar to the one identified in the yCc:CcP crystal structure.