Zhang Bixia, Kang ChulHee, Davydov Dmitri R
Department of Chemistry, Washington State University, Pullman, WA 99164, USA.
Biology (Basel). 2022 Mar 25;11(4):510. doi: 10.3390/biology11040510.
NADPH-cytochrome P450 reductase (CPR) from (SbCPR) serves as an electron donor for cytochrome P450 essential for monolignol and lignin production in this biofuel crop. The CPR enzymes undergo an ample conformational transition between the closed and open states in their functioning. This transition is triggered by electron transfer between the FAD and FMN and provides access of the partner protein to the electron-donating FMN domain. To characterize the electron transfer mechanisms in the monolignol biosynthetic pathway better, we explore the conformational transitions in SbCPR with rapid scanning stop-flow and pressure-perturbation spectroscopy. We used FRET between a pair of donor and acceptor probes incorporated into the FAD and FMN domains of SbCPR, respectively, to characterize the equilibrium between the open and closed states and explore its modulation in connection with the redox state of the enzyme. We demonstrate that, although the closed conformation always predominates in the conformational landscape, the population of open state increases by order of magnitude upon the formation of the disemiquinone state. Our results are consistent with several open conformation sub-states differing in the volume change (Δ) of the opening transition. While the Δ characteristic of the oxidized enzyme is as large as -88 mL/mol, the interaction of the enzyme with the nucleotide cofactor and the formation of the double-semiquinone state of CPR decrease this value to -34 and -18 mL/mol, respectively. This observation suggests that the interdomain electron transfer in CPR increases protein hydration, while promoting more open conformation. In addition to elucidating the functional choreography of plant CPRs, our study demonstrates the high exploratory potential of a combination of the pressure-perturbation approach with the FRET-based monitoring of protein conformational transitions.
来自[具体来源未提及]的NADPH - 细胞色素P450还原酶（CPR，即SbCPR）作为细胞色素P450的电子供体，对这种生物燃料作物中单木质醇和木质素的产生至关重要。CPR酶在其功能过程中会在关闭状态和开放状态之间经历充分的构象转变。这种转变由FAD和FMN之间的电子转移触发，并使伴侣蛋白能够接触到供电子的FMN结构域。为了更好地表征单木质醇生物合成途径中的电子转移机制，我们利用快速扫描停流和压力扰动光谱法研究了SbCPR中的构象转变。我们分别在SbCPR的FAD和FMN结构域中引入一对供体和受体探针，利用荧光共振能量转移（FRET）来表征开放状态和关闭状态之间的平衡，并探讨其与酶的氧化还原状态相关的调节作用。我们证明，尽管在构象格局中关闭构象始终占主导，但在半醌态形成时，开放状态的比例会增加几个数量级。我们的结果与几种开放构象亚状态一致，这些亚状态在开放转变的体积变化（Δ）方面有所不同。虽然氧化酶的Δ特征高达 - 88 mL/mol，但酶与核苷酸辅因子的相互作用以及CPR双半醌态的形成分别将该值降低至 - 34 mL/mol和 - 18 mL/mol。这一观察结果表明，CPR中的结构域间电子转移增加了蛋白质的水合作用，同时促进了更开放的构象。除了阐明植物CPR的功能编排外，我们的研究还证明了压力扰动方法与基于FRET的蛋白质构象转变监测相结合具有很高的探索潜力。
