Blahut Matthew R, Dawson Michael E, Kisgeropoulos Effie C, Ledinina Anastasia E, Mulder David W, King Paul W
Biosciences Center, National Renewable Energy Lab, Golden, Colorado, USA.
Biosciences Center, National Renewable Energy Lab, Golden, Colorado, USA.
J Biol Chem. 2024 Dec;300(12):107936. doi: 10.1016/j.jbc.2024.107936. Epub 2024 Oct 28.
The HoxEFUYH complex of Synechocystis PCC 6803 (S. 6803) consists of a HoxEFU ferredoxin:NAD(P)H oxidoreductase subcomplex and a HoxYH [NiFe]-hydrogenase subcomplex that catalyzes reversible H oxidation. Prior studies have suggested that the presence of HoxE is required for reactivity with ferredoxin; however, it is unknown how HoxE is functionally integrated into the electron transfer network of the HoxEFU:ferredoxin complex. Deciphering electron transfer pathways is challenged by the rich iron-sulfur cluster content of HoxEFU, which includes a [2Fe-2S] cluster in each subunit, along with multiple [4Fe-4S] clusters and a flavin cofactor. To resolve the role of HoxE, we determined the biophysical and thermodynamic properties of each [2Fe-2S] cluster in HoxEFU using steady-state and potentiometric EPR analysis in combination with square wave voltammetry (SWV). The temperature-dependence of the EPR signal for HoxE confirmed the coordination of a single [2Fe-2S] cluster that was shown by SWV to have an E = -424 mV (versus SHE). Strikingly, when the E of the HoxE [2Fe-2S] cluster was analyzed in HoxEFU titrations, it was shifted by >100 mV to an E < -525 mV (versus SHE). EPR titrations of HoxEFU gave an E value for the [2Fe-2S] cluster of HoxF, E = -419 mV and HoxU, E = -349 mV. These values were used to re-analyze the diaphorase kinetics in reactions performed with ferredoxins with varying E's. The results are formulated into a model of HoxEFU:ferredoxin reactivity and the role of HoxE in mediating electron transfer within the HoxEFU:ferredoxin complex.
集胞藻PCC 6803（S. 6803）的HoxEFUYH复合物由一个HoxEFU铁氧化还原蛋白：NAD（P）H氧化还原酶亚复合物和一个催化可逆氢氧化的HoxYH [NiFe] -氢化酶亚复合物组成。先前的研究表明，与铁氧化还原蛋白反应需要HoxE的存在；然而，尚不清楚HoxE如何在功能上整合到HoxEFU：铁氧化还原蛋白复合物的电子传递网络中。HoxEFU丰富的铁硫簇含量给解析电子传递途径带来了挑战，HoxEFU每个亚基都包含一个[2Fe-2S]簇，还有多个[4Fe-4S]簇和一个黄素辅因子。为了阐明HoxE的作用，我们结合方波伏安法（SWV），通过稳态和电位EPR分析，确定了HoxEFU中每个[2Fe-2S]簇的生物物理和热力学性质。HoxE的EPR信号的温度依赖性证实了单个[2Fe-2S]簇的配位，SWV显示其E = -424 mV（相对于标准氢电极）。引人注目的是，当在HoxEFU滴定中分析HoxE [2Fe-2S]簇的E时，它偏移了>100 mV至E < -525 mV（相对于标准氢电极）。HoxEFU的EPR滴定得出HoxF的[2Fe-2S]簇的E值为E = -419 mV，HoxU的E值为E = -349 mV。这些值被用于重新分析用具有不同E值的铁氧化还原蛋白进行的反应中的递氢酶动力学。结果被整合到一个HoxEFU：铁氧化还原蛋白反应性以及HoxE在介导HoxEFU：铁氧化还原蛋白复合物内电子传递中的作用的模型中。
