Bradley Amy L, Chobot Sarah E, Arciero David M, Hooper Alan B, Elliott Sean J
Department of Chemistry, Boston University, Boston, Massachusetts 02215, USA.
J Biol Chem. 2004 Apr 2;279(14):13297-300. doi: 10.1074/jbc.C400026200. Epub 2004 Feb 18.
Here the cytochrome c peroxidase (CcP) from Nitrosomonas europaea is examined using the technique of catalytic protein film voltammetry. Submonolayers of the bacterial diheme enzyme at a pyrolytic graphite edge electrode give catalytic, reductive signals in the presence of the substrate hydrogen peroxide. The resulting waveshapes indicate that CcP is bound non-covalently in a highly active configuration. The native enzyme has been shown to possess two heme groups of low and high potential (L and H, -260 and +450 mV versus hydrogen, respectively), and here we find that the catalytic waves of the N. europaea enzyme have a midpoint potential of >500 mV and a shape that corresponds to a 1-electron process. The signals increase in magnitude with hydrogen peroxide concentration, revealing Michaelis-Menten kinetics and K(m) = 55 microm. The midpoint potentials shift with substrate concentration, indicating the electrochemically active species observed in our data corresponds to a catalytic species. The potentials also shift with respect to pH, and the pH dependence is interpreted in terms of a two pK(a) model for proton binding. Together the data show that the electrochemistry of the N. europaea cytochrome c peroxidase is unlike other peroxidases studied to date, including other bacterial enzymes. This is discussed in terms of a catalytic model for the N. europaea enzyme and compared with other cytochrome c peroxidases.
在这里，利用催化蛋白质膜伏安法技术对来自欧洲亚硝化单胞菌的细胞色素c过氧化物酶（CcP）进行了研究。在热解石墨边缘电极上的细菌双血红素酶亚单层在底物过氧化氢存在下给出催化还原信号。所得波形表明CcP以高活性构型非共价结合。已证明天然酶具有两个低电位和高电位的血红素基团（L和H，相对于氢分别为-260和+450 mV），在这里我们发现欧洲亚硝化单胞菌酶的催化波具有>500 mV的中点电位和对应于单电子过程的形状。信号幅度随过氧化氢浓度增加，显示出米氏动力学，K(m)=55微摩尔。中点电位随底物浓度变化，表明我们数据中观察到的电化学活性物种对应于一种催化物种。电位也随pH值变化，并且根据质子结合的双pK(a)模型解释了pH依赖性。这些数据共同表明，欧洲亚硝化单胞菌细胞色素c过氧化物酶的电化学与迄今为止研究的其他过氧化物酶不同，包括其他细菌酶。本文根据欧洲亚硝化单胞菌酶的催化模型进行了讨论，并与其他细胞色素c过氧化物酶进行了比较。
