Histidine-Gated Proton-Coupled Electron Transfer to the Cu_A Site of Nitrous Oxide Reductase

Copper-containing nitrous oxide reductase (N₂OR) is the only known enzyme to catalyze the conversion of the environmentally critical greenhouse gas nitrous oxide (N₂O) to dinitrogen (N₂) as the final step of bacterial denitrification. Other than its unique tetranuclear active site Cu_Z, the binuclear electron entry point Cu_A is also utilized in other enzymes, including cytochrome c oxidase. In the Cu_A site of Pseudomonas stutzeri N₂OR, a histidine ligand was found to undergo a conformational flip upon binding of the substrate N₂O between the two copper centers. Here we report on the systematic mutagenesis and spectroscopic and structural characterization of this histidine and surrounding H-bonding residues, based on an established functional expression system for PsN₂OR in E. coli. A single hydrogen bond from Ser550 is sufficient to stabilize an unbound conformation of His583, as shown in a Asp576Ala variant, while the additional removal of the hydrogen bond in a Asp576Ala/Ser550Ala double variant compelled His583 to stay in a bound conformation as a ligand to Cu_A. Systematic mutagenesis of His583 to Ala, Asp, Asn, Glu, Gln, Lys, Phe, Tyr, and Trp showed that although both the Cu_Z and Cu_A sites were present in all the variants, only the ones with a protonable side chain, i.e., His, Asp, and Glu, were able to mediate electron transfer at physiological pH. This observation is in line with a proton-coupled electron transfer mechanism at the Cu_A site of N₂OR.