posted on 2020-12-30, 16:05authored byLin Zhang, Eckhard Bill, Peter M. H. Kroneck, Oliver Einsle
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 CuZ, 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 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 CuA. Systematic mutagenesis of His583 to Ala, Asp, Asn, Glu, Gln, Lys,
Phe, Tyr, and Trp showed that although both the CuZ and
CuA 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 CuA site of N2OR.