School of Chemistry, UNSW Sydney, NSW, Australia.
FEBS J. 2018 Aug;285(16):2972-2986. doi: 10.1111/febs.14519. Epub 2018 Jun 7.
The enzyme nitrogenase converts N to NH , and hydrogenates many other small unsaturated molecules, using multiple electrons and multiple protons. The protein contains a number of water structures in the vicinity of the active site, FeMo-co, and functional roles have been assigned to two of these with detailed mechanisms proposed for the serial ingress of protons and the egress of product NH . A separate small water pool (SWP), in a different part of the protein surrounding FeMo-co, has unknown function. A recent investigation of protein crystals soaked in low-pH buffer revealed changes in residues near this SWP, and suggested that it could be involved in proton transfer steps. This paper examines the SWP in three protein crystal structures, Azotobacter vinelandii (Av1) and Clostridium pasterianum (Cp1) in their neutral resting states, and Cp1 at low pH. The H atoms, not observed crystallographically, were patched in through density functional calculations using large protein models. Optimisation of the various possibilities, with assessment against crystal dimensions, yielded the most probable distributions of hydrogen atoms in the hydrogen bonds, and the location of H O in the low-pH state. These detailed structures vary in water content and water involvement with surrounding residues, and vary also in their hydrogen bonding to S atoms of FeMo-co. A conserved mechanism for proton transfer to FeMo-co is not evident, and it is concluded that the SWP has no role in the mechanism of nitrogenase.
酶氮还原酶将 N 转化为 NH ，并氢化许多其他不饱和小分子，使用多个电子和多个质子。该蛋白在活性位点附近含有许多水结构，FeMo-co，并为其中两个水结构赋予了功能角色，并提出了详细的质子连续进入和产物 NH 流出的机制。在 FeMo-co 周围蛋白的不同部位有一个单独的小水腔（SWP），其功能未知。最近对浸泡在低 pH 缓冲液中的蛋白晶体的研究揭示了该 SWP 附近残基的变化，并表明它可能参与质子转移步骤。本文研究了三种蛋白晶体结构中的 SWP，即固氮菌（Av1）和巴氏梭菌（Cp1）在中性静息状态下，以及 Cp1 在低 pH 下的状态。通过使用大蛋白模型的密度泛函计算，对未在晶体中观察到的 H 原子进行了修补。通过对各种可能性进行优化，并根据晶体尺寸进行评估，得出了氢键中 H 原子最可能的分布，以及低 pH 状态下 H O 的位置。这些详细的结构在含水量和与周围残基的水参与方面有所不同，并且在与 FeMo-co 的 S 原子的氢键方面也有所不同。没有明显的质子向 FeMo-co 转移的保守机制，因此得出结论，SWP 在氮还原酶的机制中没有作用。
