New insights into the reaction capabilities of His 195 adjacent to the active site of nitrogenase.

The active site of the enzyme nitrogenase is the FeMo-cofactor (FeMo-co), a C-centred Fe7 MoS9 cluster, connected to the surrounding MoFe protein via ligands Cys275 and His442 . Density functional calculations, involving 14 additional surrounding amino acids, focus on His195 because its mutation causes important reactivity changes, including almost complete loss of ability to reduce N2 to NH3 . The Nε side-chain of His195 is capable of hydrogen bonding to S2B, bridging Fe2 and Fe6 of FeMo-co, believed to be the main reaction domain of nitrogenase. Details are presented for the possible ways in which protonated or deprotonated Nε of His195 interact with S2B or S2B-H or Fe2 or Fe2-H or Fe-(H2 ). Movements of the His195 side-chain allow formation of a significant short dihydrogen bond between Nε of His195 and H on Fe2: Nε-H••H-Fe2, with H-H=1.39Å. It is shown that a 180° rotation of the imidazole ring of His195 is not able to facilitate transfer of protons from the protein surface to FeMo-co. His195 is able to move H atoms to and from S2B, and the characteristics of H transfer between S2B and Nε of His195 are described, together with their dependence on the protonation state of His195 and the redox state of FeMo-co. The water molecule on the posterior Nδ side of His195 can mediate proton transfer to and from the side-chain of Tyr228 . The accumulated results suggest that protonated His195 could be the agent for the first, most difficult, transfer of H to bound substrate N2 .