New insights into the reaction capabilities of His195 adjacent to the active site of nitrogenase.

The active site of the enzyme nitrogenase is the FeMo-cofactor (FeMo-co), a C-centred Fe7MoS9 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.