No Laughing Matter: The Unmaking of the Greenhouse Gas Dinitrogen Monoxide by Nitrous Oxide Reductase - The Metal-Driven Biogeochemistry of Gaseous Compounds in the Environment - page 190

3.2.3 Unexpected Flexibility: Cu A in P. stutzeri Nitrous
Oxide Reductase

In the structure of purple N 2 O reductase from P. stutzeri , the Cu A site differed not
only in the consistently longer metal-metal bond when compared to earlier structure
determinations, but also in the conformation of one of the coordinating ligands
to a copper, His583 [ 32 ]. While all earlier structure determinations of proteins
containing a Cu A site - natively or through engineering of the Cu A -binding loop -
invariably showed the conserved ligand environment described above, His583 of
P. stutzeri N 2 O reductase was found in two different conformations, with its N ʴ
atom either coordinating Cu 2 of the Cu A site, or forming a hydrogen bond to the
hydroxyl group of nearby residue Ser550 (Figure 6 ). The two different conforma-
tions are related through a rotation of the imidazole moiety of the side chain of
His583 by approximately 135 [ 32 ]. With its N ʵ nitrogen, His583 retains a hydro-

gen bond to the -carboxy group of Asp576, an aspartate residue located at the

surface of the enzyme, in an area that was postulated as a possible docking site for
an electron transfer partner [ 37 ]. The arrangement observed in the crystal structures
thus gave rise to the hypothesis that His583 can act as a gate for electron transfer
from an external electron donor to the Cu A site of N 2 O reductase. If a direct
coordination of His583 to Cu 2 was indeed a prerequisite for reduction of Cu A ,
then the rotation of the histidine side chain towards Ser550 would serve this conduit
and prevent the enzyme from executing its catalytic function [ 32 , 44 ].
However, in order to function as an efficient gate the flip of the imidazole
moiety should be linked to a triggering event within the catalytic cycle. In various
structures of N 2 OR form I, His583 was observed in either conformation, flipped in
or out with respect to Cu 2 , indicating a significant degree of structural flexibility on
the part of the reductase. Notably, in several crystal structure determinations
the conformation of His583 differed between the two monomers of the same
enzyme dimer. This is not straightforward to rationalize in a crystal structure that
always represents the macroscopic conformational average over all unit cells, and it
may imply a slight asymmetry of the dimeric arrangement that affects the crystal-
lization process. No such asymmetry has been described to date, and it will be
interesting to conduct an in-depth analysis based on high-quality structural data.
To date His583 (or the corresponding residue in other Cu A domains) was so far
only found in a flipped-out state in the purple form I of N 2 OR (Figure 6 ). While
Ser550 is a conserved residue in N 2 O reductase, it is absent in heme-copper
oxidases, where no flip of a histidine ligand to Cu A seems to occur. In N 2 O
reductase, the purple form I has the second metal site, Cu Z , in a [4Cu:2S] confor-
mation, so that there must be structural changes in the vicinity of both metal sites
that enable His583 to abandon its coordination to Cu 2 of Cu A . The exact determi-
nants of these changes remain to be identified, but their functional relevance is
underlined by the finding that the one case where His583 was always found flipped
towards Cu A - thus enabling electron transfer from an external partner - was when
substrate was bound to the nearby Cu Z site (see Section 5.3 ).

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