Tosha Takehiko, Yoshioka Shiro, Hori Hiroshi, Takahashi Satoshi, Ishimori Koichiro, Morishima Isao
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-8501, Japan.
Biochemistry. 2002 Nov 26;41(47):13883-93. doi: 10.1021/bi0261037.
We characterized electron transfer (ET) from putidaredoxin (Pdx) to the mutants of cytochrome P450(cam) (P450(cam)), in which one of the residues located on the putative binding site to Pdx, Gln360, was replaced with Glu, Lys, and Leu. The kinetic analysis of the ET reactions from reduced Pdx to ferric P450(cam) (the first ET) and to ferrous oxygenated P450(cam) (the second ET) showed the dissociation constants (K(m)) that were moderately perturbed for the Lys and Leu mutants and the distinctly increased for the Glu mutant. Although the alterations in K(m) indicate that Gln360 is located at the Pdx binding site, the effects of the Gln360 mutations (0.66-20-fold of that of wild type) are smaller than those of the Arg112 mutants (25-2500-fold of that of wild type) [Unno, M., et al. (1996) J. Biol. Chem. 271, 17869-17874], allowing us to conclude that Gln360 much less contributes to the complexation with Pdx than Arg112. The first ET rate (35 s(-1) for wild-type P450(cam)) was substantially reduced in the Glu mutant (5.4 s(-1)), while less perturbation was observed for the Lys (53 s(-1)) and Leu (23 s(-1)) mutants. In the second ET reaction, the retarded ET rate was detected only in the Glu mutant but not in the Lys and Leu mutants. These results showed the smaller mutational effects of Gln360 on the ET reactions than those of the Arg112 mutants. In contrast to the moderate perturbations in the kinetic parameters, the mutations at Gln360 significantly affected both the standard enthalpy and entropy of the redox reaction of P450(cam), which cause the negative shift of the redox potentials for the Fe(3+)/Fe(2+) couple by 20-70 mV. Since the amide group of Gln360 is located near the carbonyl oxygen of the amide group of the axial cysteine, it is plausible that the mutation at Gln360 perturbs the electronic interaction of the axial ligand with heme iron, resulting in the reduction of the redox potentials. We, therefore, conclude that Gln360 primarily regulates the ET reaction of P450(cam) by modulating the redox potential of the heme iron and not by the specific interaction with Pdx or the formation of the ET pathway that are proposed as the regulation mechanism of Arg112.
我们对从恶臭假单胞菌铁氧化还原蛋白（Pdx）到细胞色素P450（cam）（P450（cam））突变体的电子转移（ET）进行了表征，其中位于假定的与Pdx结合位点上的一个残基Gln360被Glu、Lys和Leu取代。对从还原态Pdx到铁离子态P450（cam）（第一次ET）以及到亚铁氧合态P450（cam）（第二次ET）的ET反应进行动力学分析，结果显示Lys和Leu突变体的解离常数（K(m)）受到适度干扰，而Glu突变体的解离常数明显增加。尽管K(m)的变化表明Gln360位于Pdx结合位点，但Gln360突变的影响（为野生型的0.66 - 20倍）小于Arg112突变体的影响（为野生型的25 - 2500倍）[Unno, M.,等人（1996年）《生物化学杂志》271, 17869 - 17874]，这使我们得出结论，与Arg112相比，Gln360对与Pdx形成复合物的贡献要小得多。第一次ET速率（野生型P450（cam）为35 s⁻¹）在Glu突变体中大幅降低（5.4 s⁻¹），而Lys（53 s⁻¹）和Leu（23 s⁻¹）突变体的干扰较小。在第二次ET反应中，仅在Glu突变体中检测到ET速率延迟，而在Lys和Leu突变体中未检测到。这些结果表明，Gln360对ET反应的突变影响比Arg112突变体小。与动力学参数的适度干扰相反，Gln360处的突变显著影响了P450（cam）氧化还原反应的标准焓和熵，这导致Fe(3+)/Fe(2+)电对的氧化还原电位负移20 - 70 mV。由于Gln360的酰胺基团位于轴向半胱氨酸酰胺基团的羰基氧附近，Gln360处的突变扰乱轴向配体与血红素铁的电子相互作用从而导致氧化还原电位降低是合理的。因此，我们得出结论，Gln360主要通过调节血红素铁的氧化还原电位来调节P450（cam）的ET反应，而不是通过与Pdx的特异性相互作用或作为Arg112调节机制所提出的ET途径的形成。
