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 181

originally studied enzyme from P. stutzeri [ 32 ]. Gaining an unambiguous picture of
the function and properties of N 2 O reductase has been complicated by the fact that
the enzyme has been isolated in various forms that differ in content and state of the
active centers, as well as in their activity.

3.1 Anatomy of an Unusual Copper Enzyme

The enzyme N 2 O reductase is the product of the nosZ gene, an open reading frame
encoding a protein of approximately 640 amino acid residues that is located in
the bacterial periplasmic space. Variations to this organization were reported for the
denitrifying hyperthermophilic crenarchaeon Pyrobaculum aerophilum [ 33 ], and
for few genera of bacteria including Thiomicrospira denitrificans and Wolinella
succinogenes that contain an additional cytochrome c domain in their nosZ gene
[ 23 ]. The two domains of the canonical NosZ protein fold into an N-terminal,
seven-bladed

-sandwich that attains the cupredoxin
fold commonly observed in mononuclear type-I copper proteins [ 34 ]. Accordingly,
each individual domain has a distinct metal center embedded, and the interaction of
two chains in the dimeric enzyme assures that they are located in an appropriate
orientation to afford a functional interaction (Figure 3 ).
NosZ proteins form stable homodimers with a strict head-to-tail arrangement,
where the N-terminal domain of one monomer interacts with the C-terminal domain
of the other and vice versa . In consequence, the metal centers within a single protein
chain are more than 40

ʲ -propeller, and a C-terminal ʲ

apart in the NosZ dimer, while the distance between
the centers in different chains amounts to a mere 10

Å

, placing it well within the
range required for efficient electron transfer [ 35 ]. Both metal centers of N 2 O
reductase exclusively employ copper, and both exhibit a degree of specialization
that is only observed here. The cupredoxin domain is the site of the binuclear Cu A
center, an electron transfer site with a characteristic spectroscopic signature that is
also found in many respiratory oxidases. Its function in N 2 O reductase is to mediate
single-electron transfer from an external electron donor, commonly a c -type cyto-
chrome or a cupredoxin, to the second metal site where substrate reduction occurs.
This second site is a tetranuclear Cu center termed Cu Z that so far has only been
found in N 2 O reductase. Its architecture and exact composition have been a matter
of debate for many years (see Section 3.3 ).

Å

3.1.1 Distinct Forms of the Enzyme

The observed differences in operon architecture are in at least one case directly
related to the structure of the NosZ protein itself. Nos operons that lack the nosR
gene and instead contain nosG , nosH , and further periplasmic cytochromes, typi-
cally contain a larger open reading frame for nosZ that includes a third domain with
the

ʱ -helical secondary structure and the typical CXXCH heme binding motif of