Lipscomb J D
Biochemistry. 1980 Jul 22;19(15):3590-9. doi: 10.1021/bi00556a027.
Cytochrome P-450cam is a low-spin Fe3+hemoprotein (g = 2.45, 2.26, and 1.91) which is made 60% high spin (g = 7.85, 3.97, and 1.78) at 12 K by the addition of 1 mol of substrate per mol of enzyme. Low-temperature EPR spectra show that the low-spin fraction of substrate-bound P-450cam contains two magnetic species. The majority species has an unusual EPR spectrum (g = 2.42, 2.24, and 1.97) which connot be simulated by using the range of crystal field parameters known for other heme proteins. The minority species has the same g values as substrate-free enzyme. Both low-spin species show Curie law temperature dependence below 50 K and have similar saturation behavior. Above 50 K the g = 2.42, 2.24, and 1.97 species rapidly loses signal intensity. The distribution of low-spin species is pH dependent (apparent pKa = 6.2) with the g = 2.42, 2.24, and 1.97 magnetic species favored at high pH. The substrate binding stoichiometry and the equilibria observed in the low-spin fraction suggest that there are not multiple protein forms of cytochrome P-450cam. Putidaredoxin and other effector molecules which specifically catalyze hydroxylation convert either the high-spin or the g = 2.42, 2.24, and 1.97 low-spin species to another new magnetic species (g = 2.47, 2.26, and 1.91). This species is only seen in the presence of substrate, and its stability reflects the catalytic potency of the effector molecule. The EPR and UV-visible spectra of cytochrome P-420 depend upon the manner in which the P-420 is generated. Incubation with acetone or reaction with N-ethylmaleimide or diethyl pyrocarbonate generates P-420 with different spectral characteristics. Through identification of active-site amino acids by chemical modification and comparison with porphyrin model complexes, the range of ligands likely to participate in each of the EPR detectable species is assigned. Mechanisms of interconversion of these species and their bearing on catalysis are discussed.
细胞色素P-450cam是一种低自旋Fe3+血红素蛋白（g = 2.45、2.26和1.91），每摩尔酶加入1摩尔底物后，在12 K时会有60%转变为高自旋（g = 7.85、3.97和1.78）。低温电子顺磁共振光谱表明，与底物结合的P-450cam的低自旋部分包含两种磁性物质。主要物质具有不寻常的电子顺磁共振光谱（g = 2.42、2.24和1.97），无法用其他血红素蛋白已知的晶体场参数范围进行模拟。次要物质的g值与无底物酶相同。两种低自旋物质在50 K以下均表现出居里定律温度依赖性，且具有相似的饱和行为。在50 K以上，g = 2.42、2.24和1.97的物质会迅速失去信号强度。低自旋物质的分布取决于pH值（表观pKa = 6.2），在高pH值下，g = 2.42、2.24和1.97的磁性物质占优势。底物结合化学计量以及在低自旋部分观察到的平衡表明，细胞色素P-450cam不存在多种蛋白质形式。特异性催化羟基化反应的腐胺铁氧还蛋白和其他效应分子会将高自旋或g = 2.42、2.24和1.97的低自旋物质转化为另一种新的磁性物质（g = 2.47、2.26和1.91）。这种物质仅在有底物存在时出现，其稳定性反映了效应分子的催化效力。细胞色素P-420的电子顺磁共振和紫外可见光谱取决于P-420的生成方式。用丙酮孵育或与N-乙基马来酰亚胺或焦碳酸二乙酯反应会生成具有不同光谱特征的P-420。通过化学修饰鉴定活性位点氨基酸并与卟啉模型配合物进行比较，确定了可能参与每种可通过电子顺磁共振检测到的物质的配体范围。讨论了这些物质相互转化的机制及其与催化作用的关系。
