Yonetani T, Anni H
J Biol Chem. 1987 Jul 15;262(20):9547-54.
Electronic absorption and electron paramagnetic resonance (EPR) spectroscopic examinations revealed that a freshly prepared cytochrome c peroxidase (CCP) contains a penta-coordinated high spin ferric protoheme group. The penta-coordinated high spin state of fresh CCP is maintained in a remarkably wide range of pH (4-8). The freezing of fresh CCP induces the reversible coordination of an internal strong field ligand to the heme iron to form a hexa-coordinated low spin compound, which shows EPR extrema at gx = 2.70, gy = 2.20 and gz = 1.78. In the presence of glycerol the freezing-induced artifacts are eliminated and the fresh enzyme exhibits an EPR spectrum of rhombically distorted axial symmetry with EPR extrema at gx = 6.4, gy = 5.3, and gz = 1.97 at 10 K, characteristic of the penta-coordinated high spin enzyme. Upon aging CCP is converted to a hexa-coordinated high spin state due to the coordination of an internal weak field ligand to the heme iron. This conversion is accelerated at acidic pH values, and its reversibility varies from fully reversible to irreversible depending on the degree of enzyme aging. The aging-induced hexa-coordinated CCP is unreactive with hydrogen peroxide and exhibits an EPR spectrum of purely axial symmetry with extrema at g = 6 and g = 2 and an electronic absorption spectrum with an intensified Soret band at 408 nm (epsilon 408 nm = 120 mM-1 cm-1) and a blue-shifted charge-transfer band at 620 nm. Spectroscopic properties of different coordination and spin states of fresh and aged CCPs are compiled in order to formulate a generalized spectroscopic characterization of penta- and hexa-coordinated high spin ferric hemoproteins.
电子吸收光谱和电子顺磁共振（EPR）光谱研究表明，新制备的细胞色素c过氧化物酶（CCP）含有一个五配位的高自旋铁原卟啉基团。新鲜CCP的五配位高自旋态在很宽的pH范围（4 - 8）内保持稳定。新鲜CCP的冷冻会诱导一个内部强场配体与血红素铁发生可逆配位，形成六配位的低自旋化合物，该化合物在gx = 2.70、gy = 2.20和gz = 1.78处呈现EPR极值。在甘油存在的情况下，冷冻诱导的假象被消除，新鲜酶在10 K时呈现出菱形畸变轴对称的EPR谱，gx = 6.4、gy = 5.3和gz = 1.97处有EPR极值，这是五配位高自旋酶的特征。随着CCP老化，由于一个内部弱场配体与血红素铁配位，它会转变为六配位高自旋态。这种转变在酸性pH值下加速，其可逆性根据酶的老化程度从完全可逆到不可逆变化。老化诱导的六配位CCP与过氧化氢不反应，呈现出纯轴对称的EPR谱，极值在g = 6和g = 2处，以及电子吸收光谱，在408 nm处有增强的Soret带（ε408 nm = 120 mM-1 cm-1），在620 nm处有蓝移的电荷转移带。为了对五配位和六配位高自旋铁血红素蛋白进行广义的光谱表征，汇编了新鲜和老化CCP不同配位和自旋态的光谱性质。
