Mei Hongkang, Geren Lois, Miller Mark A, Durham Bill, Millett Francis
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, USA.
Biochemistry. 2002 Mar 26;41(12):3968-76. doi: 10.1021/bi016020a.
The interaction of yeast iso-1-cytochrome c (yCc) with the high- and low-affinity binding sites on cytochrome c peroxidase compound I (CMPI) was studied by stopped-flow spectroscopy. When 3 microM reduced yCc(II) was mixed with 0.5 microM CMPI at 10 mM ionic strength, the Trp-191 radical cation was reduced from the high-affinity site with an apparent rate constant >3000 s(-1), followed by slow reduction of the oxyferryl heme with a rate constant of only 10 s(-1). In contrast, mixing 3 microM reduced yCc(II) with 0.5 microM preformed CMPI yCc(III) complex led to reduction of the radical cation with a rate constant of 10 s(-1), followed by reduction of the oxyferryl heme in compound II with the same rate constant. The rate constants for reduction of the radical cation and the oxyferryl heme both increased with increasing concentrations of yCc(II) and remained equal to each other. These results are consistent with a mechanism in which both the Trp-191 radical cation and the oxyferryl heme are reduced by yCc(II) in the high-affinity binding site, and the reaction is rate-limited by product dissociation of yCc(III) from the high-affinity site with apparent rate constant k(d). Binding yCc(II) to the low-affinity site is proposed to increase the rate constant for dissociation of yCc(III) from the high-affinity site in a substrate-assisted product dissociation mechanism. The value of k(d) is <5 s(-1) for the 1:1 complex and >2000 s(-1) for the 2:1 complex at 10 mM ionic strength. The reaction of horse Cc(II) with CMPI was greatly inhibited by binding 1 equiv of yCc(III) to the high-affinity site, providing evidence that reduction of the oxyferryl heme involves electron transfer from the high-affinity binding site rather than the low-affinity site. The effects of CcP surface mutations on the dissociation rate constant indicate that the high-affinity binding site used for the reaction in solution is the same as the one identified in the yCcCcP crystal structure.
通过停流光谱法研究了酵母同工酶-1-细胞色素c（yCc）与细胞色素c过氧化物酶化合物I（CMPI）上高亲和力和低亲和力结合位点的相互作用。当在10 mM离子强度下将3 microM还原型yCc（II）与0.5 microM CMPI混合时，色氨酸-191自由基阳离子从高亲和力位点被还原，表观速率常数>3000 s(-1)，随后氧合铁血红素缓慢还原，速率常数仅为10 s(-1)。相比之下，将3 microM还原型yCc（II）与0.5 microM预先形成的CMPIyCc（III）复合物混合，导致自由基阳离子以10 s(-1)的速率常数被还原，随后化合物II中的氧合铁血红素以相同的速率常数被还原。自由基阳离子和氧合铁血红素还原的速率常数均随yCc（II）浓度的增加而增加，且彼此保持相等。这些结果与一种机制一致，即色氨酸-191自由基阳离子和氧合铁血红素在高亲和力结合位点均被yCc（II）还原，且反应受yCc（III）从高亲和力位点的产物解离限制，表观速率常数为k(d)。在底物辅助产物解离机制中，将yCc（II）结合到低亲和力位点可提高yCc（III）从高亲和力位点解离的速率常数。在10 mM离子强度下，1:1复合物的k(d)值<5 s(-1)，2:1复合物的k(d)值>2000 s(-1)。将1当量的yCc（III）结合到高亲和力位点极大地抑制了马细胞色素c（Cc II）与CMPI的反应，这表明氧合铁血红素的还原涉及从高亲和力结合位点而非低亲和力位点的电子转移。细胞色素c过氧化物酶（CcP）表面突变对解离速率常数的影响表明，溶液中用于反应的高亲和力结合位点与yCcCcP晶体结构中确定的位点相同。
