Hahm S, Miller M A, Geren L, Kraut J, Durham B, Millett F
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville 72701.
Biochemistry. 1994 Feb 15;33(6):1473-80. doi: 10.1021/bi00172a025.
The reactions of recombinant cytochrome c peroxidase [CcP(MI)] and a number of CcP(MI) mutants with native and ruthenium-labeled horse ferrocytochrome c have been studied by stopped-flow spectroscopy and laser flash photolysis. At 100 mM ionic strength, pH 7.5, native horse ferrocytochrome c reduces the radical on the indole group of Trp-191 in cytochrome c peroxidase compound I (CMPI) with a second-order rate constant of 1.3 x 10(8) M-1 s-1. Ferrocytochrome c then reduces the oxyferryl heme Fe(IV) in CMPII with a rate constant of 2.0 x 10(6) M-1 s-1. The rate constant for the reduction of the radical is nearly independent of pH from 5 to 8, but the rate constant for reduction of the oxyferryl heme Fe(IV) increases 33-fold as the pH is decreased from 8 to 5. This increase in rate is correlated with the pH dependence of the electron transfer equilibrium between the radical and the oxyferryl heme Fe(IV) in the transient form of CMPII. The second-order rate constants for reduction of the radical and the oxyferryl heme in the mutants Y39F, Y42F, H181G, W223F, and Y229F are nearly the same as for wild-type CcP(MI). The intracomplex rate constants for reduction of the radical in these mutants by the ruthenium-labeled cytochrome c derivatives are also similar to that for CcP(MI). This rules out a direct role for these aromatic residues in electron transfer.(ABSTRACT TRUNCATED AT 250 WORDS)
通过停流光谱法和激光闪光光解研究了重组细胞色素c过氧化物酶[CcP(MI)]以及一些CcP(MI)突变体与天然和钌标记的马亚铁细胞色素c的反应。在100 mM离子强度、pH 7.5条件下，天然马亚铁细胞色素c以1.3×10⁸ M⁻¹ s⁻¹的二级速率常数还原细胞色素c过氧化物酶化合物I（CMPI）中色氨酸-191吲哚基团上的自由基。然后亚铁细胞色素c以2.0×10⁶ M⁻¹ s⁻¹的速率常数还原CMPII中的氧合铁血红素Fe(IV)。自由基还原的速率常数在pH 5至8范围内几乎与pH无关，但随着pH从8降至5，氧合铁血红素Fe(IV)还原的速率常数增加了33倍。该速率的增加与CMPII瞬态形式中自由基与氧合铁血红素Fe(IV)之间电子转移平衡的pH依赖性相关。Y39F、Y42F、H181G、W223F和Y229F突变体中自由基和氧合铁血红素还原的二级速率常数与野生型CcP(MI)几乎相同。这些突变体中钌标记的细胞色素c衍生物还原自由基的复合体内速率常数也与CcP(MI)相似。这排除了这些芳香族残基在电子转移中的直接作用。（摘要截短于250字）
