Koh Moonjoo, Meyer Terry E, De Smet Lina, Van Beeumen Jozef J, Cusanovich Michael A
Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ 85721, USA.
Arch Biochem Biophys. 2003 Feb 15;410(2):230-7. doi: 10.1016/s0003-9861(02)00694-x.
Steady-state kinetics for the reaction of Rhodobacter capsulatus bacterial cytochrome c peroxidase (BCCP) with its substrate cytochrome c(2) were investigated. The Rb. capsulatus BCCP is dependent on calcium for activation as previously shown for the Pseudomonas aeruginosa BCCP and Paracoccus denitrificans enzymes. Furthermore, the activity shows a bell-shaped pH dependence with optimum at pH 7.0. Enzyme activity is greatest at low ionic strength and drops off steeply as ionic strength increases, resulting in an apparent interaction domain charge product of -13. All cytochromes c(2) show an asymmetric distribution of surface charge, with a concentration of 14 positive charges near the exposed heme edge of Rb. capsulatus c(2) which potentially may interact with approximately 6 negative charges, localized near the edge of the high-potential heme of the Rb. capsulatus BCCP. To test this proposal, we constructed charge reversal mutants of the 14 positively charged residues located on the front face of Rb. capsulatus cytochrome c(2) and examined their effect on steady-state kinetics with BCCP. Mutated residues in Rb. capsulatus cytochrome c(2) that showed the greatest effects on binding and enzyme activity are K12E, K14E, K54E, K84E, K93E, and K99E, which is consistent with the site of electron transfer being located at the heme edge. We conclude that a combination of long-range, nonspecific electrostatic interactions as well as localized salt bridges between, e.g., cytochrome c(2) K12, K14, K54, and K99 with BCCP D194, D241, and D6, account for the observed kinetics.
对荚膜红细菌细菌细胞色素c过氧化物酶（BCCP）与其底物细胞色素c₂的反应进行了稳态动力学研究。如先前对铜绿假单胞菌BCCP和反硝化副球菌酶所显示的那样，荚膜红细菌BCCP的激活依赖于钙。此外，该活性表现出钟形的pH依赖性，在pH 7.0时达到最佳。酶活性在低离子强度下最大，随着离子强度增加而急剧下降，导致表观相互作用域电荷产物为-13。所有细胞色素c₂都显示出表面电荷的不对称分布，在荚膜红细菌c₂暴露的血红素边缘附近有14个正电荷集中，这可能与大约6个负电荷相互作用，这些负电荷位于荚膜红细菌BCCP高电位血红素边缘附近。为了验证这一假设，我们构建了荚膜红细菌细胞色素c₂正面14个带正电荷残基的电荷反转突变体，并研究了它们对与BCCP的稳态动力学的影响。荚膜红细菌细胞色素c₂中对结合和酶活性影响最大的突变残基是K12E、K14E、K54E、K84E、K93E和K99E，这与电子转移位点位于血红素边缘一致。我们得出结论，长程、非特异性静电相互作用以及例如细胞色素c₂的K12、K14、K54和K99与BCCP的D194、D241和D6之间的局部盐桥共同作用，解释了观察到的动力学现象。
