Abstract：
Metal-dependent, nicotine adenine dinucleotide (NAD+)-dependent formate dehydrogenases (FDHs) are complex metalloenzymes coupling biochemical transformations through intricate electron transfer pathways. Rhodobacter capsulatus FDH is a model enzyme for understanding coupled catalysis, in that reversible CO2 reduction and formate oxidation are linked to a flavin mononuclotide (FMN)-bound diaphorase module via seven iron-sulfur (FeS) clusters as a dimer of heterotetramers. Catalysis occurs at a bis-metal-binding pterin (Mo) binding two molybdopterin guanine dinucleotides (bis-MGD), a protein-based Cys residue and a participatory sulfido ligand. Insights regarding the proposed electron transfer mechanism between the bis-MGD and the FMN have been complicated by the discovery that an alternative pathway might occur via intersubunit electron transfer between two [4Fe4S] clusters within electron transfer distance. To clarify this difference, the redox potentials of the bis-MGD and the FeS clusters were determined via redox titration by EPR spectroscopy. Redox potentials for the bis-MGD cofactor and five of the seven FeS clusters could be assigned. Furthermore, substitution of the active site residue Lys295 with Ala resulted in altered enzyme kinetics, primarily due to a more negative redox potential of the A1 [4Fe4S] cluster. Finally, characterization of the monomeric FdsGBAD heterotetramer exhibited slightly decreased formate oxidation activity and similar iron-sulfur clusters reduced relative to the dimeric heterotetramer. Comparison of the measured redox potentials relative to structurally defined FeS clusters support a mechanism by which electron transfer occurs within a heterotetrameric unit, with the interfacial [4Fe4S] cluster serving as a structural component toward the integrity of the heterodimeric structure to drive efficient catalysis.
摘要：
依赖金属，尼古丁腺嘌呤二核苷酸（NAD）依赖性甲酸脱氢酶（FDHs）是复杂的金属酶，通过复杂的电子转移途径偶联生化转化。荚膜红杆菌FDH是一种用于理解偶联催化的模型酶，在这种可逆的CO2还原和甲酸氧化中，通过七个铁硫（FeS）簇作为异四聚体的二聚体与黄素单核苷酸（FMN）结合的黄递酶模块相关联。催化发生在双金属结合蝶呤（Mo）结合两个钼蝶呤鸟嘌呤二核苷酸（bis-MGD），基于蛋白质的Cys残基和参与式硫化物配体。关于bis-MGD和FMN之间提出的电子转移机制的见解由于发现可能通过电子转移距离内的两个[4Fe4S]团簇之间的亚基间电子转移而发生替代途径而变得复杂。为了澄清这种差异，bis-MGD和FeS簇的氧化还原电位通过氧化还原滴定通过EPR光谱法测定。可以分配bis-MGD辅因子和七个FeS簇中的五个的氧化还原电位。此外，用Ala取代活性位点残基Lys295导致酶动力学改变，主要是由于A1[4Fe4S]团簇的氧化还原电位更负。最后,单体FdsGBAD异四聚体的表征显示，相对于二聚体异四聚体，甲酸氧化活性略有降低，铁硫簇减少。相对于结构定义的FeS团簇测量的氧化还原电位的比较支持在异四聚体单元内发生电子转移的机制。界面[4Fe4S]团簇充当朝向异二聚结构完整性的结构组分，以驱动有效的催化。
