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首页> 外文期刊>Biochemistry >Characterization of Four Covalently-Linked Yeast Cytochrome c/Cytochrome c Peroxidase Complexes: Evidence for Electrostatic Interaction between Bound Cytochrome c Molecules
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Characterization of Four Covalently-Linked Yeast Cytochrome c/Cytochrome c Peroxidase Complexes: Evidence for Electrostatic Interaction between Bound Cytochrome c Molecules

机译：四个共价链接的酵母细胞色素c /细胞色素c过氧化物酶复合物的表征：绑定的细胞色素c分子之间的静电相互作用的证据。

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Four covalent complexes between recombinant yeast cytochrome c and cytochrome c peroxidase (rCcP) were synthesized via disulfide bond formation using specifically designed protein mutants (Papa,H.S.,and Poulos,T.L.(1995) Biochemistry 34,6573-6580).One of the complexes,designated V5C/ K79C,has cysteine residues replacing valine-5 in rCcP and lysine-79 in cytochrome c with disulfide bond formation between these residues linking the two proteins.The V5C/K79C complex has the covalently bound cytochrome c located on the back-side of cytochrome c peroxidase,approx 180~(deg) from the primary cytochrome obinding site as defined by the crystallographic structure of the 1:1 noncovalent complex (Pelletier,H.,and Kraut J.(1992) Science 258,1748-1755).Three other complexes have the covalently bound cytochrome c located approx 90~(deg) from the primary binding site and are designated K12C/K79C,N78C/ K79C,and K264C/K79C,respectively.Steady-state kinetic studies were used to investigate the catalytic properties of the covalent complexes at both 10 and 100 mM ionic strength at pH 7.5.All four covalent complexes have catalytic activities similar to those of rCcP (within a factor of 2).A comprehensive study of the ionic strength dependence of the steady-state kinetic properties of the V5C/K79C complex provides evidence for significant electrostatic repulsion between the two cytochromes bound in the 2:1 complex at low ionic strength and shows that the electrostatic repulsion decreases as the ionic strength of the buffer increases.
机译：重组酵母细胞色素c和细胞色素c过氧化物酶（rCcP）之间的四个共价复合物是使用专门设计的蛋白质突变体通过二硫键形成合成的（Papa，HS，and Poulos，TL（1995）Biochemistry 34,6573-6580）。命名为V5C / K79C的半胱氨酸残基取代了细胞色素c中的rCcP中的缬氨酸5和赖氨酸79，并且在这两个蛋白之间的两个残基之间形成了二硫键。V5C/ K79C复合物具有共价结合的细胞色素c位于细胞色素C过氧化物酶的一面，离主要细胞色素的结合位点约180°（度），由1：1非共价复合物的晶体结构定义（Pelletier，H.，and Kraut J.（1992）Science 258,1748-1755 ）。其他三个复合物的共价结合细胞色素c位于主要结合位点约90°处，分别命名为K12C / K79C，N78C / K79C和K264C / K79C。使用稳态动力学研究催化的在pH值为7.5时离子强度为10和100 mM时的共价配合物的性质。所有四种共价配合物的催化活性均与rCcP相似（在2倍以内）。对稳态离子强度依赖性的综合研究V5C / K79C络合物的动力学特性为低离子强度下结合在2：1络合物中的两种细胞色素之间的显着静电排斥提供了证据，并表明静电排斥随着缓冲液离子强度的增加而降低。

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