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 (N-2) as the final step of bacterial denitrification. Other than its unique tetranuclear active site Cu-Z, the binuclear electron entry point CUA is also utilized in other enzymes, including cytochrome c oxidase. In the CUA 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 PsN(2)OR in E. coli. A single hydrogen bond from Ser550 is sufficient to stabilize an unbound conformation of His583, as shown in a AspS76Ala 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.