School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
Faraday Discuss. 2024 Sep 11;252(0):188-207. doi: 10.1039/d4fd00020j.
The electrochemical leaf enables the electrification and control of multi-enzyme cascades by exploiting two discoveries: (i) the ability to electrify the photosynthetic enzyme ferredoxin NADP reductase (FNR), driving it to catalyse the interconversion of NADP/NADPH whilst it is entrapped in a highly porous, metal oxide electrode, and (ii) the evidence that additional enzymes can be co-entrapped in the electrode pores where, through one NADP(H)-dependent enzyme, extended cascades can be driven by electrical connection to FNR, NADP(H) recycling. By changing a critical active-site tyrosine to serine, FNR's exclusivity for NADP(H) is swapped for unphosphorylated NAD(H). Here we present an electrochemical study of this variant FNR, and show that in addition to the intended inversion of cofactor preference, this change to the active site has altered FNR's tuning of the flavin reduction potential, making it less reductive. Exploiting the ability to monitor the variant's activity with NADP(H) as a function of potential has revealed a trapped intermediate state, relieved only by applying a negative overpotential, which allows catalysis to proceed. Inhibition by NADP (very tightly bound) with respect to NAD(H) turnover was also revealed and interestingly, this inhibition changes depending on the applied potential. These findings are of critical importance for future exploitation of the electrochemical leaf.
(i) 能够使光合作用酶铁氧还蛋白 NADP 还原酶（FNR）电活化，驱动其在高多孔金属氧化物电极中催化 NADP/NADPH 的相互转化，以及 (ii) 额外的酶可以共捕获在电极孔中，通过一个 NADP(H)依赖性酶，通过与 FNR 的电连接和 NADP(H)循环，可以驱动扩展级联。通过将关键活性位点酪氨酸变为丝氨酸，FNR 对 NADP(H)的专一性被未磷酸化的 NAD(H)取代。在这里，我们对这种变体 FNR 进行了电化学研究，结果表明，除了预期的辅因子偏好反转外，这种活性位点的改变还改变了 FNR 对黄素还原电位的调谐，使其还原性降低。利用 NADP(H)作为电位函数监测变体的活性的能力揭示了一种被捕获的中间状态，只有施加负过电位才能缓解这种状态，从而允许催化反应进行。还揭示了 NADP 对 NAD(H)周转的抑制作用（非常紧密结合），有趣的是，这种抑制作用取决于施加的电位。这些发现对电化学叶的未来开发具有至关重要的意义。
