Vitello L B, Erman J E, Miller M A, Mauro J M, Kraut J
Department of Chemistry, Northern Illinois University, DeKalb 60115.
Biochemistry. 1992 Nov 24;31(46):11524-35. doi: 10.1021/bi00161a034.
The spectroscopic properties of a mutant cytochrome c peroxidase, in which Asp-235 has been replaced by an asparagine residue, were examined in both nitrate and phosphate buffers between pH 4 and 10.5. The spin state of the enzyme is pH dependent, and four distinct spectroscopic species are observed in each buffer system: a predominantly high-spin Fe(III) species at pH 4, two distinct low-spin forms between pH 5 and 9, and the denatured enzyme above pH 9.3. The spectrum of the mutant enzyme at pH 4 is dependent upon specific ion effects. Increasing the pH above 5 converts the mutant enzyme to a predominantly low-spin hydroxy complex. Subsequent conversion to a second low-spin form is essentially complete at pH 7.5. The second low-spin form has the distal histidine, His-52, coordinated to the heme iron. To evaluate the effect of the changes in coordination state upon the reactivity of the enzyme, the reaction between hydrogen peroxide and the mutant enzyme was also examined as a function of pH. The reaction of CcP(MI,D235N) with peroxide is biphasic. At pH 6, the rapid phase of the reaction can be attributed to the bimolecular reaction between hydrogen peroxide and the hydroxy-ligated form of the mutant enzyme. Despite the hexacoordination of the heme iron in this form, the bimolecular rate constant is approximately 22% that of pentacoordinate wild-type yeast cytochrome c peroxidase. The bimolecular reaction of the mutant enzyme with peroxide exhibits the same pH dependence in nitrate-containing buffers that has been described for the wild-type enzyme, indicating a loss of reactivity with the protonation of a group with an apparent pKa of 5.4. This observation eliminates Asp-235 as the source for this heme-linked ionization and strengthens the hypothesis that the pKa of 5.4 is associated with His-52. The slower phase of the reaction between peroxide and the mutant enzyme saturates at high peroxide concentration and is attributed to conversion of unreactive to reactive forms of the enzyme. The fraction of enzyme which reacts via the slow phase is dependent upon both pH and specific ion effects.
研究了一种突变型细胞色素c过氧化物酶（其中天冬氨酸-235被天冬酰胺残基取代）在pH值4至10.5的硝酸盐和磷酸盐缓冲液中的光谱性质。酶的自旋态取决于pH值，并且在每个缓冲系统中观察到四种不同的光谱种类：在pH值4时主要是高自旋Fe(III)种类，在pH值5至9之间有两种不同的低自旋形式，以及在pH值9.3以上的变性酶。突变酶在pH值4时的光谱取决于特定离子效应。将pH值提高到5以上会使突变酶转化为主要是低自旋羟基配合物。随后在pH值7.5时基本上完全转化为第二种低自旋形式。第二种低自旋形式中，远端组氨酸His-52与血红素铁配位。为了评估配位状态变化对酶反应性的影响，还研究了过氧化氢与突变酶之间的反应作为pH值的函数。CcP(MI,D235N)与过氧化物的反应是双相的。在pH值6时，反应的快速阶段可归因于过氧化氢与突变酶的羟基配位形式之间的双分子反应。尽管这种形式的血红素铁是六配位的，但双分子速率常数约为五配位野生型酵母细胞色素c过氧化物酶的22%。突变酶与过氧化物的双分子反应在含硝酸盐的缓冲液中表现出与野生型酶相同的pH依赖性，表明与表观pKa为5.4的基团质子化相关的反应性丧失。这一观察结果排除了天冬氨酸-235作为这种血红素连接电离的来源，并强化了pKa为5.4与His-52相关的假设。过氧化物与突变酶之间反应的较慢阶段在高过氧化物浓度下达到饱和，并归因于酶从不反应形式转化为反应形式。通过慢相反应的酶的比例取决于pH值和特定离子效应。
