Zoppellaro Giorgio, Harbitz Espen, Kaur Ravinder, Ensign Amy A, Bren Kara L, Andersson K Kristoffer
Department of Molecular Biosciences, University of Oslo, Post Office Box 1041 Blindern, Oslo NO-0316, Norway.
J Am Chem Soc. 2008 Nov 19;130(46):15348-60. doi: 10.1021/ja8033312. Epub 2008 Oct 24.
Cytochromes of the c type with histidine-methionine (His-Met) heme axial ligation play important roles in electron-transfer reactions and in enzymes. In this work, two series of cytochrome c mutants derived from Pseudomonas aeruginosa (Pa c-551) and from the ammonia-oxidizing bacterium Nitrosomonas europaea (Ne c-552) were engineered and overexpressed. In these proteins, point mutations were induced in a key residue (Asn64) near the Met axial ligand; these mutations have a considerable impact both on heme ligand-field strength and on the Met orientation and dynamics (fluxionality), as judged by low-temperature electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectra. Ne c-552 has a ferric low-spin (S = 1/2) EPR signal characterized by large g anisotropy with g(max) resonance at 3.34; a similar large g(max) value EPR signal is found in the mitochondrial complex III cytochrome c1. In Ne c-552, deletion of Asn64 (NeN64Delta) changes the heme ligand field from more axial to rhombic (small g anisotropy and g(max) at 3.13) and furthermore hinders the Met fluxionality present in the wild-type protein. In Pa c-551 (g(max) at 3.20), replacement of Asn64 with valine (PaN64V) induces a decrease in the axial strain (g(max) at 3.05) and changes the Met configuration. Another set of mutants prepared by insertion (ins) and/or deletion (Delta) of a valine residue adjacent to Asn64, resulting in modifications in the length of the axial Met-donating loop (NeV65Delta, NeG50N/V65Delta, PaN50G/V65ins), did not result in appreciable alterations of the originally weak (Ne c-552) or very weak (Pa c-551) axial field but had an impact on Met orientation, fluxionality, and relaxation dynamics. Comparison of the electronic fingerprints in the overexpressed proteins and their mutants reveals a linear relationship between axial strain and average paramagnetic heme methyl shifts, irrespective of Met orientation or dynamics. Thus, for these His-Met axially coordinated Fe(III), the large g(max) value EPR signal does not represent a special case as is observed for bis-His axially coordinated Fe(III) with the two His planes perpendicular to each other.
具有组氨酸 - 甲硫氨酸（His - Met）血红素轴向配位的c型细胞色素在电子转移反应和酶中发挥着重要作用。在这项工作中，构建并过量表达了源自铜绿假单胞菌（Pa c - 551）和氨氧化细菌欧洲亚硝化单胞菌（Ne c - 552）的两个系列的细胞色素c突变体。在这些蛋白质中，在靠近甲硫氨酸轴向配体的一个关键残基（Asn64）处诱导了点突变；根据低温电子顺磁共振（EPR）和核磁共振（NMR）光谱判断，这些突变对血红素配体场强度以及甲硫氨酸的取向和动力学（流动性）都有相当大的影响。Ne c - 552具有一个高铁低自旋（S = 1/2）的EPR信号，其特征是具有大的g各向异性，g（max）共振在3.34处；在线粒体复合物III细胞色素c1中也发现了类似的大g（max）值的EPR信号。在Ne c - 552中，Asn64的缺失（NeN64Delta）将血红素配体场从更轴向变为菱形（小的g各向异性，g（max）在3.13处），并且进一步阻碍了野生型蛋白质中存在的甲硫氨酸流动性。在Pa c - 551（g（max）在3.20处）中，用缬氨酸取代Asn64（PaN64V）导致轴向应变降低（g（max）在3.05处）并改变了甲硫氨酸的构型。通过在与Asn64相邻处插入（ins）和/或缺失（Delta）缬氨酸残基制备的另一组突变体，导致轴向甲硫氨酸供体环长度的改变（NeV65Delta、NeG50N/V65Delta、PaN
