A defining characteristic of bacterial cytochromes (cyt's) in the P460 family is an unusual cross-link connecting the heme porphyrin to the side chain of a lysyl residue in the protein backbone. Here, via proteomics of the periplasmic fraction of the ammonia-oxidizing bacterium (AOB) Nitrosomonas europaea, we report the identification of a variant member of the P460 family that contains a methionyl residue in place of the cross-linking lysine. We formally designate this protein cytochrome "c'β-Met" to distinguish it from other members bearing different residues at this position (e.g., cyt c'β-Phe from the methane-oxidizing Methylococcus capsulatus Bath). As isolated, the monoheme cyt c'β-Met is high-spin (S = 5/2). Optical spectroscopy suggests that a cross-link is absent. Hydroxylamine, the substrate for the cross-linked cyt P460 from N. europaea, did not appreciably alter the optical spectrum of cyt c'β with up to 1000-fold excess at pH 7.5. Cyt c'β-Met did however bind 1 equiv of H2O2, and with a slight excess, Mössbauer spectroscopy indicated the formation of a semistable ferryl (FeIV═O) Compound II-like species. The corresponding electron paramagnetic resonance showed a very low intensity signal indicative of a radical at g = 2.0. Furthermore, cyt c'β-Met exhibited guaiacol-dependent peroxidase activity (kcat = 20.0 ± 1.2 s-1; KM = 2.6 ± 0.4 mM). Unlike cyt c'β-Met, cyt P460 showed evidence of heme inactivation in the presence of 2 equiv of H2O2 with no appreciable guaiacol-dependent peroxidase activity. Mutagenesis of the cross-linking lysyl residue to an alanine in cyt P460, however, reversed this lack of activity.

中文翻译：

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细胞色素c'β-Met是P460缺乏血红素-赖氨酰交联的超家族的变体：过氧化物酶模拟物生成Ferryl中间体。

P460家族中细菌细胞色素（cyt's）的定义特征是将血红素卟啉连接到蛋白质骨架中赖氨酰残基的侧链的异常交联。在这里，我们通过氨氧化细菌（Nitrosomonas europaea）的周质组分的蛋白质组学，报告了鉴定出含有甲硫酰基残基代替交联赖氨酸的P460家族变异成员。我们正式指定此蛋白质细胞色素“c'β-Met”，以使其与在此位置带有不同残基的其他成员区分开（例如，来自甲烷氧化甲基囊球菌浴的cytc'β-Phe）。如孤立的，单血红素cytc'β-Met是高自旋的（S = 5/2）。光谱学表明不存在交联。羟胺 来自欧洲猪笼草的交联的cyt P460的底物在pH 7.5时并没有明显地改变cytc'β的光谱，其过量高达1000倍。然而，Cytc'β-Met确实结合了1当量的H2O2，并且稍有过量，Mössbauer光谱表明形成了半稳定的Ferryl（FeIV = O）化合物II类物质。相应的电子顺磁共振显示出非常低的强度信号，表明在g = 2.0处存在自由基。此外，cytc'β-Met表现出依赖愈创木酚的过氧化物酶活性（kcat = 20.0±1.2 s-1； KM = 2.6±0.4 mM）。与cytc'β-Met不同，cyt P460在存在2当量的H2O2的情况下显示血红素失活的证据，没有明显的愈创木酚依赖性过氧化物酶活性。然而，cyt P460中赖氨酸残基与丙氨酸交联的诱变作用，