Electron paramagnetic resonance detectable states of cytochrome P-450cam

作者：John D. Lipscomb · 发表于：Biochemistry · 年份：1980 · DOI：10.1021/bi00556a027 · 被引用次数：179 · 研究领域：Electron Spin Resonance Studies、Spectroscopy and Quantum Chemical Studies、Photosynthetic Processes and Mechanisms

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 t...