Vitello L B, Erman J E, Miller M A, Wang J, Kraut J
Department of Chemistry, Northern Illinois University, DeKalb 60115.
Biochemistry. 1993 Sep 21;32(37):9807-18. doi: 10.1021/bi00088a036.
The crystallographic structures of two cytochrome c peroxidase (CcP) mutants, CcP(R48L) and CcP(R48K), have been determined. In addition, the electronic absorption spectrum and the hydrogen peroxide reactivity of these two mutants have been determined between pH 4 and 8. Both the crystallographic structure and the electronic absorption spectrum of CcP(R48L) are consistent with exclusive pentacoordination of the heme iron between pH 4 and 6.5. At higher pH, CcP(R48L) forms an alkaline bis-imidazole form of CcP with the distal histidine coordinated to the heme iron. The apparent pKA for this transition is 7.5 in CcP(R48L). The observed pseudo-first-order rate constant for the reaction between CcP(R48L) and hydrogen peroxide saturates at high peroxide concentrations. The data are consistent with a rate-limiting oxygen-oxygen bond scission at high peroxide concentrations. The observed rate of the bond scission step ranges between 1000 and 1950 s-1, an estimated 2 orders of magnitude slower than for wild-type enzyme. The data suggest that the protonated form of His-52 increases the bond scission step by a factor of 2. The properties of the CcP(R48K) mutant are significantly different from those of CcP(R48L). The crystal structure of CcP(R48K) shows Lys-48 occupying the putative peroxide binding site. The electronic absorption spectrum indicates that CcP(R48K) is predominantly pentacoordinate at neutral pH but with detectable amounts of hexacoordinate forms. Two ionizable groups affect the electronic absorption spectrum of CcP(R48K). An apparent ionization near pH 4 produces an enzyme with increased hexacoordination, while an apparent pKA of 6.9 generates the alkaline bis-imidazole form. The peroxide reaction saturates at high peroxide concentrations for CcP(R48K) and is attributed to a conformational-gating mechanism. The maximum rate for the reaction between CcP(R48K) and hydrogen peroxide is probably limited by the movement of either Lys-48 or His-52. This rate is 200 and 290 s-1 in nitrate-containing buffers and phosphate buffers, respectively. Evidence is provided that Arg-48 in wild-type enzyme is responsible for nitrate binding in the heme pocket and for stabilizing CcP Compound I.
已测定了两种细胞色素c过氧化物酶（CcP）突变体CcP（R48L）和CcP（R48K）的晶体结构。此外，还测定了这两种突变体在pH值4至8之间的电子吸收光谱和过氧化氢反应活性。CcP（R48L）的晶体结构和电子吸收光谱均表明，在pH值4至6.5之间，血红素铁仅为五配位。在较高pH值下，CcP（R48L）形成CcP的碱性双咪唑形式，远端组氨酸与血红素铁配位。在CcP（R48L）中，这种转变的表观pKA为7.5。在高过氧化氢浓度下，CcP（R48L）与过氧化氢反应的观测到的准一级速率常数达到饱和。这些数据与高过氧化氢浓度下限速的氧-氧键断裂一致。观测到的键断裂步骤的速率在1000至1950 s-1之间，估计比野生型酶慢约2个数量级。数据表明，His-52的质子化形式使键断裂步骤加快了2倍。CcP（R48K）突变体的性质与CcP（R48L）的性质显著不同。CcP（R48K）的晶体结构显示Lys-48占据了假定的过氧化物结合位点。电子吸收光谱表明，CcP（R48K）在中性pH值下主要为五配位，但有可检测量的六配位形式。两个可电离基团影响CcP（R48K）的电子吸收光谱。在pH值4附近的表观电离产生六配位增加的酶，而表观pKA为6.9时产生碱性双咪唑形式。对于CcP（R48K），在高过氧化氢浓度下过氧化物反应达到饱和，这归因于构象门控机制。CcP（R48K）与过氧化氢反应的最大速率可能受Lys-48或His-52移动的限制。在含硝酸盐缓冲液和磷酸盐缓冲液中，该速率分别为200和290 s-1。有证据表明，野生型酶中的Arg-48负责血红素口袋中的硝酸盐结合并稳定CcP化合物I。
