Stemp E D, Hoffman B M
Department of Chemistry, Northwestern University, Evanston, Illinois 60208-3113.
Biochemistry. 1993 Oct 12;32(40):10848-65. doi: 10.1021/bi00091a041.
We have studied the affinity and stoichiometry of binding of cytochrome c (Cc) to zinc-substituted cytochrome c peroxidase [(ZnP)CcP], which is structurally and electrostatically equivalent to ferrous CcP. Transient absorption spectroscopy has been used to measure both the total quenching of the triplet-state (ZnP)CcP [3(ZnP)CcP] by Fe3+Cc and the fraction of that quenching that is due to electron transfer (et). This redox quenching results in the formation of an intermediate (I) containing the zinc porphyrin pi-cation radical [(ZnP)+CcP] and Fe2+Cc. In titrations of (ZnP)CcP with Fe3+Cc(F) at low ionic strength, where F represents the fungal cytochromes c from Candida krusei, Pichia membranefaciens, or the yeast protein iso-1, the appearance of the et intermediate lags behind the total quenching, with appreciable formation of I occurring only for Cc to CcP ratios > 1. This behavior results from the formation of a 2:1 complex, where one Fe3+Cc(F) binds to a high-affinity domain that exhibits strong quenching yet is et-inactive, while the second Fe3+Cc(F) binds to a low-affinity domain that allows efficient et quenching. At constant concentrations of both proteins, raising the ionic strength eliminates most of the et quenching but reduces the total quenching only minimally, confirming that et occurs preferentially at the low-affinity binding domain, which is the more sensitive to ionic strength. Analogous experiments also favor a 2:1 binding stoichiometry for horse Cc [Cc(horse)] at low ionic strength, with et quenching again proceeding much more favorably in the 2:1 complex than in the 1:1 complex, as with Cc(F). However, the Fe3+Cc(horse) quenches only by electron transfer, unlike the Cc(F). The decay of the triplet-state (ZnP)CcP or magnesium-substituted CcP [(MgP)CcP] was examined during titrations with Fe3+Cc to determine limits for the dissociation rate constant (koff) for the complex. Fe3+Cc(horse) bound to the high-affinity domain in a 1:1 complex at low ionic strength is in rapid exchange, with koff > 50 S-1, whereas Fe3+Cc(F) has koff < 200 s-1. Both types of Fe3+Cc have koff > 10(4)S-1 when they are bound to the low-affinity domain in a 2:1 complex, at both low and high ionic strengths. In contrast, when in the ferrous form, both types of Cc have much lower values of koff (< 10 S-1) at low ionic strength when bound to the low-affinity domain.(ABSTRACT TRUNCATED AT 400 WORDS)
我们研究了细胞色素c（Cc）与锌取代的细胞色素c过氧化物酶[（ZnP）CcP]结合的亲和力和化学计量关系，（ZnP）CcP在结构和静电方面与亚铁细胞色素c过氧化物酶（CcP）相当。瞬态吸收光谱已用于测量Fe3 + Cc对三重态（ZnP）CcP [3（ZnP）CcP]的总猝灭以及由于电子转移（et）导致的猝灭部分。这种氧化还原猝灭导致形成一种中间体（I），其包含锌卟啉π - 阳离子自由基[（ZnP）+ CcP]和Fe2 + Cc。在低离子强度下用Fe3 + Cc（F）滴定（ZnP）CcP时，其中F代表来自克鲁斯假丝酵母、膜醭毕赤酵母的真菌细胞色素c或酵母蛋白iso - 1，et中间体的出现滞后于总猝灭，仅当Cc与CcP的比例> 1时才会有明显的I形成。这种行为是由2:1复合物的形成导致的，其中一个Fe3 + Cc（F）与一个高亲和力结构域结合，该结构域表现出强烈的猝灭但无et活性，而第二个Fe3 + Cc（F）与一个低亲和力结构域结合，该结构域允许有效的et猝灭。在两种蛋白质浓度恒定的情况下，提高离子强度可消除大部分et猝灭，但仅略微降低总猝灭，这证实et优先发生在对离子强度更敏感的低亲和力结合结构域。类似的实验也支持在低离子强度下马Cc [Cc（马）]的2:1结合化学计量关系，与Cc（F）一样，et猝灭在2:1复合物中比在1:1复合物中进行得更有利。然而，与Cc（F）不同，Fe3 + Cc（马）仅通过电子转移进行猝灭。在用Fe3 + Cc滴定期间，检查了三重态（ZnP）CcP或镁取代的CcP [（MgP）CcP]的衰减，以确定复合物解离速率常数（koff）的极限。在低离子强度下，与高亲和力结构域以1:1复合物结合的Fe3 + Cc（马）处于快速交换状态，koff> 50 s - 1，而Fe3 + Cc（F）的koff <200 s - 1。当两种类型的Fe3 + Cc在2:1复合物中与低亲和力结构域结合时，在低离子强度和高离子强度下，koff均> 10（4）s - 1。相反，当处于亚铁形式时，两种类型的Cc在低离子强度下与低亲和力结构域结合时，koff值要低得多（<10 s - 1）。（摘要截断于400字）
