What is the role of the isolated small water pool near FeMo-co, the active site of nitrogenase?

Abstract

The enzyme nitrogenase converts N2 to NH3, 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 NH3. 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 H3O+ 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.