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 191

3.3 The Tetranuclear Cu Z Center

In contrast to Cu A , the second metal site in N 2 O reductase, Cu Z , has no precedence
in any other known enzyme, and its exact architecture and relevant states remain to
be fully understood. As the addition of the strong reductant sodium dithionite,
Na 2 S 2 O 4 , produced a blue species with an absorption maximum around 640 nm and
a distinct EPR spectrum, the presence of a second copper site in N 2 OR was
suspected early on [ 26 ]. A copper site that was not reduced by dithionite seemed
highly unusual, and as copper determinations at the time yielded on average 8 Cu
per enzyme dimer [ 41 , 42 ], the site - now designated Cu Z - was considered to be a
second dinuclear center with a sulfide ligand that gave rise to the characteristic
charge-transfer band [ 74 ]. The EPR signature of Cu Z , with g || ¼

2.18 and g ⊥ ¼ 2.06,

was distinct from the signature of Cu A (Figure 4 ) and remained after the latter was
reduced to an all-Cu(I), diamagnetic state [ 43 ]. This form of Cu Z was described as
an S resting state of the cluster [ 75 ]. In retrospect, many studies on Cu Z

suffered from the intrinsic structural and electronic heterogeneity found in Cu Z ,
and only with the advent of three-dimensional structures for different forms of
nitrous oxide reductase the picture has become clearer.
A recurring theme in literature on N 2 OR is on the different states of the Cu Z site.
After the initial description of two forms, a catalytically active Cu Z and a cataly-
tically inactive Cu Z * [ 74 , 76 , 77 ], more recent data have led to a more differentiated
(and complicated) picture. Ascorbate reduces the Cu A site to a colorless and
EPR-silent [Cu + :Cu + ] state, so that Cu Z can be straightforwardly analyzed without
influence from the other cluster. Here, the difference between both forms of Cu Z is
readily apparent. In electron excitation spectroscopy, Cu Z * shows the single LMCT
band at 640 nm described above, while the Cu Z center is characterized by
two distinct bands at 550 nm and 650 nm. Further reduction of Cu Z with sodium
dithionite depletes the absorption at 550 nm, but notably a complete reduction of
the site is not achieved and the band at 650 nm is retained even after prolonged
incubation with strong reductants. In contrast, dithionite in combination with the
redox mediator methyl viologen can reduce Cu Z * to an all-cuprous, colorless state.
After reduction of Cu A , both Cu Z and Cu Z * appear blue in color, and according
to the original assignment both species would be classified as form III N 2 OR.
Nevertheless the key to understanding the reactivity of nitrous oxide reductase
most likely lies exactly within the distinction of these states.

¼½

3.3.1 Structural Data on Cu Z

After the initial clarification of the central ligand of Cu Z to be a partially occupied
sulfide rather than on oxide [ 27 , 28 ], the Cu Z centers of M. hydrocarbonoclasticus
[ 27 ], P. denitrificans [ 30 ], and A. cycloclastes [ 31 ] were consistently described as
[4Cu:

-S] centers [ 44 , 78 ]. The picture was amended with the structure of purple
form I N 2 OR from P. stutzeri that showed a [4Cu:2S] configuration (Figure 7 ) and

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