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 184

In this form II, the 7-line hyperfine pattern in the EPR spectrum of Cu A (Figure 4 )
was generally less well resolved in the g || region, and at the time this was ascribed
to an underlying signal of a distinct form of Cu Z , termed Cu Z * [ 40 , 43 ]. As such,
Cu Z was thus associated with an active state, Cu Z * with an inactive state of the
center. Anoxic reduction of Cu A with a 10-fold molar excess of dithionite changed
the color of the protein to blue (form III), leading to a catalytically inactive state
dominated by a single charge-transfer band with a maximum at 650 nm, and an
axial S

EPR signal without a resolved hyperfine structure in the g || region
[ 40 ]. With Cu A in the reduced state, the spectroscopic features of form III solely
originate from the Cu Z center, but while a distinction was made for the EPR data, it
was not initially recognized that the UV/vis signatures reveal the identity and ratio
of Cu Z versus Cu Z * that reflect on the structure/function relationship of the
enzyme's active site (Figure 5 )[ 44 ].

¼½

Figure 5 Electron excitation spectra of N 2 O reductase. (a) The spectrum of purple N 2 OR (form I)
shows maxima at 538 nm and 795 nm and can be deconvoluted into individual bands by a multi-
Gaussian fit. (b) The individual contributions of the Cu A and Cu Z centers can be reconstructed
from the fit, so that the properties of the two sites can be studied in isolation. In addition, the
reduction with sodium ascorbate will only reduce Cu A , and the Cu Z contribution is readily
accessible experimentally. (c)InN 2 OR form I, the contribution of the Cu Z site can be modelled
by two bands with maxima at 552 nm and 650 nm. Further reduction with sodium dithionite will
only deplete the transition at 552 nm, while the remaining band persists. (d) In form II N 2 OR, Cu Z
is present in the [4Cu:S] Cu Z * state, with only a single band with a maximum at 650 nm from the
tetranuclear cluster. Contrary to form I, this state can be further reduced with sodium dithionite,
yielding an enzyme that is all-Cu(I) and thus colorless.