Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston, MA, 02215, USA.
Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA.
J Biol Inorg Chem. 2019 Sep;24(6):889-898. doi: 10.1007/s00775-019-01701-1. Epub 2019 Aug 28.
The formate dehydrogenase enzyme from Cupriavidus necator (FdsABG) carries out the two-electron oxidation of formate to CO, but is also capable of reducing CO back to formate, a potential biofuel. FdsABG is a heterotrimeric enzyme that performs this transformation using nine redox-active cofactors: a bis(molybdopterin guanine dinucleotide) (bis-MGD) at the active site coupled to seven iron-sulfur clusters, and one equivalent of flavin mononucleotide (FMN). To better understand the pathway of electron flow in FdsABG, the reduction potentials of the various cofactors were examined through direct electrochemistry. Given the redundancy of cofactors, a truncated form of the FdsA subunit was developed that possesses only the bis-MGD active site and a singular [4Fe-4S] cluster. Electrochemical characterization of FdsABG compared to truncated FdsA shows that the measured reduction potentials are remarkably similar despite the truncation with two observable features at - 265 mV and - 455 mV vs SHE, indicating that the voltammetry of the truncated enzyme is representative of the reduction potentials of the intact heterotrimer. By producing truncated FdsA without the necessary maturation factors required for bis-MGD insertion, a form of the truncated FdsA that possesses only the [4Fe-4S] was produced, which gives a single voltammetric feature at - 525 mV, allowing the contributions of the molybdenum cofactor to be associated with the observed feature at - 265 mV. This method allowed for the deconvolution of reduction potentials for an enzyme with highly complex cofactor content to know more about the thermodynamic landscape of catalysis.
铜绿假单胞菌（Cupriavidus necator）中的甲酸脱氢酶（FdsABG）能够将甲酸进行两电子氧化为 CO，但也能够将 CO 还原回甲酸，这是一种潜在的生物燃料。FdsABG 是一种杂三聚体酶，使用九个氧化还原活性辅因子进行这种转化：活性位点的一个双（钼喋呤鸟嘌呤二核苷酸）（bis-MGD）与七个铁硫簇结合，以及一个黄素单核苷酸（FMN）当量。为了更好地了解 FdsABG 中电子流的途径，通过直接电化学法检查了各种辅因子的还原电位。鉴于辅因子的冗余性，开发了 FdsA 亚基的截断形式，该形式仅具有 bis-MGD 活性位点和一个单个[4Fe-4S]簇。与截断 FdsA 相比，FdsABG 的电化学特性表明，尽管截短，但测量的还原电位非常相似，有两个可观察到的特征在-265 mV 和-455 mV 相对于 SHE，表明截短酶的伏安法代表完整杂三聚体的还原电位。通过不生产具有 bis-MGD 插入所需的成熟因子的截断 FdsA，生产出仅具有[4Fe-4S]的截断 FdsA 的形式，在-525 mV 处给出单个伏安特征，允许将钼辅因子的贡献与观察到的-265 mV 处的特征相关联。该方法允许对具有高度复杂辅因子含量的酶的还原电位进行去卷积，以更深入地了解催化的热力学景观。
