School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane 4072, Australia.
Department of Plant Biology, Technische Universitaet, Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany.
Biochim Biophys Acta Bioenerg. 2021 Mar 1;1862(3):148358. doi: 10.1016/j.bbabio.2020.148358. Epub 2020 Dec 24.
Nitrate reductase (NR) from the fungus Neurospora crassa is a complex homodimeric metallo-flavoenzyme, where each protomer contains three distinct domains; the catalytically active terminal molybdopterin cofactor, a central heme-containing domain, and an FAD domain which binds with the natural electron donor NADPH. Here, we demonstrate the catalytic voltammetry of variants of N. crassa NRs on a modified Au electrode with the electrochemically reduced forms of benzyl viologen (BV) and anthraquinone sulfonate (AQS) acting as artificial electron donors. The biopolymer chitosan used to entrap NR on the electrode non-covalently and the enzyme film was both stable and highly active. Electrochemistry was conducted on two distinct forms; one lacking the FAD cofactor and the other lacking both the FAD and heme cofactors. While both enzymes showed catalytic nitrate reductase activity, removal of the heme cofactor resulted in a more significant effect on the rate of nitrate reduction. Electrochemical simulation was carried out to enable kinetic characterisation of both the NR:nitrate and NR:mediator reactions.
来自粗糙脉孢菌的硝酸盐还原酶（NR）是一种复杂的同二聚体金属黄素酶，每个亚基包含三个不同的结构域；催化活性末端钼喋呤辅因子、中央含血红素的结构域和与天然电子供体 NADPH 结合的 FAD 结构域。在这里，我们在修饰的 Au 电极上展示了粗糙脉孢菌 NR 变体的催化伏安法，其中电化学还原的苄基紫精（BV）和蒽醌磺酸盐（AQS）作为人工电子供体。用于非共价固定 NR 的生物聚合物壳聚糖和酶膜既稳定又具有高活性。电化学在两种不同的形式上进行；一种缺乏 FAD 辅因子，另一种缺乏 FAD 和血红素辅因子。虽然两种酶都显示出催化硝酸盐还原酶的活性，但去除血红素辅因子对硝酸盐还原的速率有更显著的影响。进行了电化学模拟，以实现 NR：硝酸盐和 NR：介体反应的动力学特征化。
