Rodríguez-Marañón M J, Qiu F, Stark R E, White S P, Zhang X, Foundling S I, Rodríguez V, Schilling C L, Bunce R A, Rivera M
Department of Chemistry, Oklahoma State University, Stillwater 74078, USA.
Biochemistry. 1996 Dec 17;35(50):16378-90. doi: 10.1021/bi961895o.
The role played by the outer mitochondrial membrane (OM) cytochrome b5 heme propionate groups in the electrostatic binding between OM cytochrome b5 and horse heart cytochrome c was investigated by 13C NMR spectroscopy and X-ray crystallography. To achieve these aims, 13C-labeled heme OM cytochrome b5 was expressed in Escherichia coli as previously described [Rivera M., Walker, F.A. (1995) Anal. Biochem. 230, 295-302]. Assignment of the resonances arising from the heme propionate carbons in ferricytochrome b5 was carried out by a combination of one- and two-dimensional NMR experiments. Titrations of [13C]heme-labeled OM cytochrome b5 with horse heart cytochrome c were carried out in order to monitor the resonances arising from the heme propionate carbonyl carbons in OM cytochrome b5. The results from these titrations clearly show that only the heme propionate located on the exposed heme edge in OM cytochrome b5 participates in the electrostatic stabilization of the complex between OM cytochrome b5 and horse heart cytochrome c. Similar experiments carried out monitoring 13C resonances arising from several other heme substituents demonstrated that the stoichiometry of the complex is 1:1. A conditional binding constant, K which equals 3.8 x 10(4) +/- 1.4 x 10(4) at mu = 0.02 M, was obtained for the formation of the complex by fitting the binding curves obtained experimentally to a model based on this stoichiometry. The X-ray crystal structure of rat liver OM cytochrome b5 solved to 2.7 A resolution shows that the structures of bovine liver microsomal cytochrome b5 and rat liver OM cytochrome b5 are almost identical when compared at medium resolution. The similarity between the two structures, combined with the findings that only the heme propionate located on the exposed heme edge of OM cytochrome b5 participates in the electrostatic binding to cytochrome c and that the stability of this complex is similar to that measured for the association between microsomal cytochrome b5 and cytochrome c, clearly indicates that the site of interaction on OM cytochrome b5 is almost identical to the one elucidated for microsomal cytochrome b5. It is therefore possible to conclude that the large body of information gathered by many investigators for the nonphysiological interaction between microsomal cytochrome b5 and cytochrome c (recently reviewed) [Mauk, A. G. Mauk, M. R., Moore, G. R., & Northrup, S. H. (1995) Bioenerg. Biomembr. 27, 311-330] has indeed biological as well as pedagogical validity.
通过13C核磁共振光谱法和X射线晶体学研究了线粒体外膜（OM）细胞色素b5血红素丙酸基团在OM细胞色素b5与马心细胞色素c之间静电结合中所起的作用。为实现这些目标，如先前所述[Rivera M., Walker, F.A.（1995年）《分析生物化学》230，295 - 302]，在大肠杆菌中表达了13C标记血红素的OM细胞色素b5。通过一维和二维核磁共振实验相结合的方法，对高铁细胞色素b5中血红素丙酸碳产生的共振进行了归属。用马心细胞色素c对[13C]血红素标记的OM细胞色素b5进行滴定，以监测OM细胞色素b5中血红素丙酸羰基碳产生的共振。这些滴定结果清楚地表明，只有位于OM细胞色素b5暴露血红素边缘的血红素丙酸参与了OM细胞色素b5与马心细胞色素c之间复合物的静电稳定作用。对由其他几个血红素取代基产生的13C共振进行监测的类似实验表明，该复合物的化学计量比为1:1。通过将实验获得的结合曲线拟合到基于该化学计量比的模型中，得到了该复合物形成的条件结合常数K，在μ = 0.02 M时，K等于3.8×10(4)±1.4×10(4)。解析到2.7 Å分辨率的大鼠肝脏OM细胞色素b5的X射线晶体结构表明，在中等分辨率下比较时，牛肝微粒体细胞色素b5和大鼠肝脏OM细胞色素b5的结构几乎相同。这两种结构之间的相似性，再加上只有位于OM细胞色素b5暴露血红素边缘的血红素丙酸参与与细胞色素c的静电结合以及该复合物的稳定性与微粒体细胞色素b5和细胞色素c之间缔合所测得的稳定性相似这一发现，清楚地表明OM细胞色素b5上的相互作用位点与微粒体细胞色素b5所阐明的位点几乎相同。因此可以得出结论，许多研究人员收集的关于微粒体细胞色素b5和细胞色素c之间非生理性相互作用的大量信息（最近有综述）[Mauk, A. G., Mauk, M. R., Moore, G. R., & Northrup, S. H.（1995年）《生物能量与生物膜》27，311 - 330]确实具有生物学和教学方面的有效性。
