Effect of arginine-48 replacement on the reaction between cytochrome c peroxidase and hydrogen peroxide.
Effect of arginine-48 replacement on the reaction between cytochrome c peroxidase and hydrogen peroxide.
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DOI：
10.1021/bi00088a036
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发表时间：
1993-09
期刊：
影响因子：
2.9
通讯作者：
L. Vitello;J. Erman;Mark A Miller;Jimin Wang;J. Kraut
中科院分区：
文献类型：
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作者：
L. Vitello;J. Erman;Mark A Miller;Jimin Wang;J. Kraut
测定了两个细胞色素c过氧化物酶（CcP）突变体CcP（R48 L）和CcP（R48 K）的晶体结构。此外，这两个突变体的电子吸收光谱和过氧化氢的反应性已被确定之间的pH 4和8。CcP（R48 L）的晶体结构和电子吸收光谱与pH 4和6.5之间的血红素铁的排他性五配位一致。在较高pH下，CcP（R48 L）形成CcP的碱性双咪唑形式，其中远端组氨酸与血红素铁配位。在CcP（R48 L）中，该转变的表观pKa为7.5。所观察到的伪一级速率常数CcP（R48 L）和过氧化氢之间的反应在高过氧化物浓度饱和。这些数据是一致的，在高过氧化物浓度的限速氧-氧键断裂。观察到的键断步骤的速率范围在1000和1950 s-1之间，估计比野生型酶慢2个数量级。数据表明，质子化形式的His-52使键断裂步骤增加2倍。CcP（R48 K）突变体的性质与CcP（R48 L）突变体的性质显著不同。CcP（R48 K）的晶体结构显示Lys-48占据推定的过氧化物结合位点。电子吸收光谱表明，CcP（R48 K）主要是五配位在中性pH值，但与可检测量的六配位形式。CcP（R48 K）的电子吸收光谱受两个可电离基团的影响。pH 4附近的表观电离产生具有增加的六配位的酶，而6.9的表观pKA产生碱性双咪唑形式。过氧化物反应饱和在高过氧化物浓度的CcP（R48 K），是由于构象门控机制。CcP（R48 K）和过氧化氢之间反应的最大速率可能受到Lys-48或His-52运动的限制。该速率在含硝酸盐缓冲液和磷酸盐缓冲液中分别为200和290 s-1。提供了野生型酶中的Arg-48负责血红素口袋中的硝酸盐结合和稳定CcP化合物I的证据。
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.