Felice Alfons K G, Schuster Christian, Kadek Alan, Filandr Frantisek, Laurent Christophe V F P, Scheiblbrandner Stefan, Schwaiger Lorenz, Schachinger Franziska, Kracher Daniel, Sygmund Christoph, Man Petr, Halada Petr, Oostenbrink Chris, Ludwig Roland
Biocatalysis and Biosensing Research Group, Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
BIOCEV-Institute of Microbiology, The Czech Academy of Sciences, Prumyslova 595, 252 50 Vestec, Czech Republic.
ACS Catal. 2021 Jan 15;11(2):517-532. doi: 10.1021/acscatal.0c05294. Epub 2020 Dec 24.
The natural function of cellobiose dehydrogenase (CDH) to donate electrons from its catalytic flavodehydrogenase (DH) domain via its cytochrome (CYT) domain to lytic polysaccharide monooxygenase (LPMO) is an example of a highly efficient extracellular electron transfer chain. To investigate the function of the CYT domain movement in the two occurring electron transfer steps, two CDHs from the ascomycete (CDHIIA and CDHIIB) and five chimeric CDH enzymes created by domain swapping were studied in combination with the fungus' own LPMOs (LPMO9C and LPMO9F). Kinetic and electrochemical methods and hydrogen/deuterium exchange mass spectrometry were used to study the domain movement, interaction, and electron transfer kinetics. Molecular docking provided insights into the protein-protein interface, the orientation of domains, and binding energies. We find that the first, interdomain electron transfer step from the catalytic site in the DH domain to the CYT domain depends on steric and electrostatic interface complementarity and the length of the protein linker between both domains but not on the redox potential difference between the FAD and heme cofactors. After CYT reduction, a conformational change of CDH from its closed state to an open state allows the second, interprotein electron transfer (IPET) step from CYT to LPMO to occur by direct interaction of the -type heme and the type-2 copper center. Chimeric CDH enzymes favor the open state and achieve higher IPET rates by exposing the heme cofactor to LPMO. The IPET, which is influenced by interface complementarity and the heme redox potential, is very efficient with bimolecular rates between 2.9 × 10 and 1.1 × 10 M s.
纤维二糖脱氢酶（CDH）通过其细胞色素（CYT）结构域将电子从其催化黄素脱氢酶（DH）结构域转移至裂解多糖单加氧酶（LPMO），这一自然功能是高效细胞外电子传递链的一个实例。为了研究CYT结构域运动在两个电子传递步骤中的功能，对来自子囊菌的两种CDH（CDHIIA和CDHIIB）以及通过结构域交换产生的五种嵌合CDH酶与该真菌自身的LPMO（LPMO9C和LPMO9F）进行了联合研究。采用动力学和电化学方法以及氢/氘交换质谱来研究结构域运动、相互作用和电子转移动力学。分子对接提供了关于蛋白质-蛋白质界面、结构域取向和结合能的见解。我们发现，从DH结构域中的催化位点到CYT结构域的第一步结构域间电子转移取决于空间和静电界面互补性以及两个结构域之间蛋白质连接子的长度，而不取决于FAD和血红素辅因子之间的氧化还原电位差。CYT还原后，CDH从其关闭状态转变为开放状态的构象变化使得第二步蛋白质间电子转移（IPET）步骤，即从CYT到LPMO，通过 -型血红素和2型铜中心的直接相互作用而发生。嵌合CDH酶有利于开放状态，并通过将血红素辅因子暴露于LPMO来实现更高的IPET速率。受界面互补性和血红素氧化还原电位影响的IPET非常高效，双分子速率在2.9×10至1.1×10 M s之间。
