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

Copper-containing nitrous oxide reductase (NOR) is the only known enzyme to catalyze the conversion of the environmentally critical greenhouse gas nitrous oxide (NO) to dinitrogen (N) as the final step of bacterial denitrification. Other than its unique tetranuclear active site Cu, the binuclear electron entry point Cu is also utilized in other enzymes, including cytochrome oxidase. In the Cu site of NOR, a histidine ligand was found to undergo a conformational flip upon binding of the substrate NO 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 NOR in . 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. Systematic mutagenesis of His583 to Ala, Asp, Asn, Glu, Gln, Lys, Phe, Tyr, and Trp showed that although both the Cu and Cu 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 site of NOR.