Bacterial formation of trimethylamine (TMA) from carnitine in the gut microbiome has been linked to cardiovascular disease. During this process, the two-component carnitine monooxygenase (CntAB) catalyzes the oxygen-dependent cleavage of carnitine to TMA and malic semialdehyde. Individual redox states of the reductase CntB and the catalytic component CntA were investigated based on mutagenesis and electron paramagnetic resonance (EPR) spectroscopic approaches. Protein ligands of the flavin mononucleotide (FMN) and the plant-type [2Fe-2S] cluster of CntB and also of the Rieske-type [2Fe-2S] cluster and the mononuclear [Fe] center of CntA were identified. EPR spectroscopy of variant CntA proteins suggested a hierarchical metallocenter maturation, Rieske [2Fe-2S] followed by the mononuclear [Fe] center. NADH-dependent electron transfer via the redox components of CntB and within the trimeric CntA complex for the activation of molecular oxygen was investigated. EPR experiments indicated that the two electrons from NADH were allocated to the plant-type [2Fe-2S] cluster and to FMN in the form of a flavin semiquinone radical. Single-turnover experiments of this reduced CntB species indicated the translocation of the first electron onto the [Fe] center and the second electron onto the Rieske-type [2Fe-2S] cluster of CntA to finally allow for oxygen activation as a basis for carnitine cleavage. EPR spectroscopic investigation of CntA variants indicated an unusual intermolecular electron transfer between the subunits of the CntA trimer via the “bridging” residue Glu-205. On the basis of these data, a redox catalytic cycle for carnitine monooxygenase was proposed.

中文翻译：

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人体肠道中的肉碱代谢：鲍曼不动杆菌的双组分肉碱单加氧酶 CntAB 的表征。

肠道微生物群中肉碱细菌形成的三甲胺 (TMA) 与心血管疾病有关。在此过程中，双组分肉碱单加氧酶 (CntAB) 催化肉碱依赖氧裂解为 TMA 和苹果酸半醛。基于诱变和电子顺磁共振 (EPR) 光谱方法研究了还原酶 CntB 和催化组分 CntA 的各个氧化还原状态。鉴定了黄素单核苷酸 (FMN) 和 CntB 的植物型 [2Fe-2S] 簇以及 Rieske 型 [2Fe-2S] 簇和 CntA 的单核 [Fe] 中心的蛋白质配体。变异 CntA 蛋白的 EPR 光谱表明分层金属中心成熟，Rieske [2Fe-2S] 紧随其后的是单核 [Fe] 中心。研究了通过 CntB 的氧化还原成分和在三聚体 CntA 复合物中激活分子氧的 NADH 依赖性电子转移。EPR 实验表明，来自 NADH 的两个电子以黄素半醌自由基的形式分配给植物型 [2Fe-2S] 簇和 FMN。这种还原的 CntB 物种的单周转实验表明，第一个电子易位到 [Fe] 中心，第二个电子易位到 CntA 的 Rieske 型 [2Fe-2S] 簇，最终允许氧活化作为肉碱的基础解理。CntA 变体的 EPR 光谱研究表明，CntA 三聚体的亚基之间通过“桥接”残基 Glu-205 进行了不寻常的分子间电子转移。在这些数据的基础上，