Download MendeleyHistidine-Gated Proton-Coupled Electron Transfer to the Cu A Site of Nitrous Oxide Reductase.
Zhang, L., Bill, E., Kroneck, P.M.H., Einsle, O.(2021) J Am Chem Soc 143: 830-838
- PubMed: 33377777
- DOI: https://doi.org/10.1021/jacs.0c10057
- Primary Citation of Related Structures:
7APY, 7AQ0, 7AQ2, 7AQ3, 7AQ4, 7AQ5, 7AQ6, 7AQ7, 7AQ8, 7AQ9 - PubMed Abstract:
Copper-containing nitrous oxide reductase (N 2 OR) is the only known enzyme to catalyze the conversion of the environmentally critical greenhouse gas nitrous oxide (N 2 O) 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 Cu A is also utilized in other enzymes, including cytochrome c oxidase. In the Cu A site of Pseudomonas stutzeri N 2 OR, a histidine ligand was found to undergo a conformational flip upon binding of the substrate N 2 O 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 Ps N 2 OR 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 N 2 OR.
Organizational Affiliation:
Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, Albertstraße 21, 79104 Freiburg im Breisgau, Germany.
