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 196

very low redox potentials in vitro , and it additionally required the presence of an
abiological redox mediator. Both components will not be readily available in the
oxidizing environment of the bacterial periplasm, and to date there is no mechanism
known to drive such low-potential redox chemistry outside the cytoplasm in an
in vivo situation [ 44 ]. Second, the most careful isolations of N 2 OR, in particular
those in the absence of dioxygen, commonly yield a high Cu Z /Cu Z * ratio, i.e., a high
proportion of the tetranuclear site in the [4Cu:2S] state [ 32 , 40 , 47 ]. It thus seems
safe to assume that the Cu Z * center is only generated from Cu Z through the loss of a
sulfur. N 2 OR is isolated from its natural host and from cells that have turned over
N 2 O in a physiological context. If indeed the [4Cu:2S] state was an inactive one,
this would imply that the bacteria have a way to remove the S 2 sulfide anion from
Cu Z for reductive activation and catalysis, but to subsequently replenish the center
to its [4Cu:2S] form that then can be isolated. The biogenesis of CuZ is not fully
understood to date (see Section 4.3 ), but it has long been known that there is no
straightforward way to re-introduce sulfur into a defective Cu Z center.
4 Biogenesis and Assembly of Nitrous Oxide Reductase
Denitrification is a respiratory metabolism that relies on the generation of a proton
motive force across the cytoplasmic membrane. All its enzymatic steps are located
in the periplasm (in the case of Gram-negative denitrifiers), and the required
metalloenzymes therefore must be assembled in this compartment that in several
aspects differs from the 'inside' of the cell, the cytoplasm. First, the assembly of
metal centers in biomolecules is never an unspecific process. This is particularly
true for the relatively rare and highly toxic copper that is tightly chaperoned at all
times in a physiological context [ 84 ]. Denitrifiers thus require not only the different
enzymes, but in most cases also a complex biogenesis pathway that assembles the
active sites post-translationally. Second, the general currency of energy for the cell,
ATP, is not available in the periplasm, so that any energy-consuming step in said
pathways must be fueled in a different way.
4.1 The nos Operon
The structural gene for N 2 O reductase is termed nosZ [ 85 ] (Figure 8 ). In most
instances, the derived NosZ protein shows the two-domain architecture
mentioned above (Figure 3 ), but in some cases an additional domain encoded for
a c -type cytochrome that presumably represents a fused electron transfer partner
(see Section 3.1 )[ 23 , 78 ]. This may not represent a fundamental difference in the
functionality of the enzymes, but it has substantial consequences for the required
machinery for N 2 OR biogenesis. Two-domain NosZ proteins are exported via the