Kracher Daniel, Zahma Kawah, Schulz Christopher, Sygmund Christoph, Gorton Lo, Ludwig Roland
Department of Food Sciences and Technology, Food Biotechnology Laboratory, University of Natural Resources and Life Sciences, Vienna, Austria.
Department of Analytical Chemistry, Biochemistry and Structural Biology, Lund University, Sweden.
FEBS J. 2015 Aug;282(16):3136-48. doi: 10.1111/febs.13310. Epub 2015 May 16.
The flavocytochrome cellobiose dehydrogenase (CDH) is secreted by wood-decomposing fungi, and is the only known extracellular enzyme with the characteristics of an electron transfer protein. Its proposed function is reduction of lytic polysaccharide mono-oxygenase for subsequent cellulose depolymerization. Electrons are transferred from FADH2 in the catalytic flavodehydrogenase domain of CDH to haem b in a mobile cytochrome domain, which acts as a mediator and transfers electrons towards the active site of lytic polysaccharide mono-oxygenase to activate oxygen. This vital role of the cytochrome domain is little understood, e.g. why do CDHs exhibit different pH optima and rates for inter-domain electron transfer (IET)? This study uses kinetic techniques and docking to assess the interaction of both domains and the resulting IET with regard to pH and ions. The results show that the reported elimination of IET at neutral or alkaline pH is caused by electrostatic repulsion, which prevents adoption of the closed conformation of CDH. Divalent alkali earth metal cations are shown to exert a bridging effect between the domains at concentrations of > 3 mm, thereby neutralizing electrostatic repulsion and increasing IET rates. The necessary high ion concentration, together with the docking results, show that this effect is not caused by specific cation binding sites, but by various clusters of Asp, Glu, Asn, Gln and the haem b propionate group at the domain interface. The results show that a closed conformation of both CDH domains is necessary for IET, but the closed conformation also increases the FAD reduction rate by an electron pulling effect.
黄素细胞色素纤维二糖脱氢酶（CDH）由木材分解真菌分泌，是唯一已知的具有电子传递蛋白特性的细胞外酶。其推测功能是还原裂解多糖单加氧酶，以便随后进行纤维素解聚。电子从CDH催化黄素脱氢酶结构域中的FADH2转移到移动细胞色素结构域中的血红素b，该细胞色素结构域充当介质并将电子转移到裂解多糖单加氧酶的活性位点以激活氧。细胞色素结构域的这一重要作用鲜为人知，例如，为什么CDH在域间电子转移（IET）方面表现出不同的最适pH值和速率？本研究使用动力学技术和对接来评估两个结构域的相互作用以及由此产生的关于pH值和离子的IET。结果表明，在中性或碱性pH值下报道的IET消除是由静电排斥引起的，这阻止了CDH采用封闭构象。二价碱土金属阳离子在浓度>3 mM时显示出在结构域之间发挥桥接作用，从而中和静电排斥并提高IET速率。所需的高离子浓度以及对接结果表明，这种效应不是由特定的阳离子结合位点引起的，而是由结构域界面处的Asp、Glu、Asn、Gln和血红素b丙酸基团的各种簇引起的。结果表明，CDH两个结构域的封闭构象对于IET是必要的，但封闭构象也通过电子拉动效应提高了FAD还原速率。
