The Production of Ammonia by Multiheme Cytochromes c - The Metal-Driven Biogeochemistry of Gaseous Compounds in the Environment - page 227

5 Other Multiheme Cytochromes c

Several interesting variations on the theme of multiheme cytochromes c were
recently discovered, as reviewed by Simon and colleagues [ 23 ] (see also [ 95 ] for
a phylogenetic model of relevant enzymes within the MCC family). Besides the
pentaheme cytochrome c NrfA, ammonia production was also reported for purified
(i) octaheme cytochrome c tetrathionate reductase (Otr enzyme 2.5 in Table 1 ),
(ii) octaheme cytochrome c nitrite reductase (Onr, enzyme 2.6 in Table 1 ), and (iii)
hydroxylamine oxidoreductase (Hao) from nitrifiers. In addition, ammonia produc-
tion from nitrite is also conceivable to occur in some ammonifying Epsilonproteo-
bacteria that usually lack a nrfA gene but encode an Hao-type octaheme cytochrome
c , tentatively named

Hao (enzyme 2.7 in Table 1 )[ 22 , 23 ].
The octaheme enzyme from S. oneidensis , originally described as tetrathionate
reductase, was structurally characterized and shown to reduce NO 2 and H 2 NOH to
NH 4 [ 96 , 97 ]. The structures of two Onr enzymes from Thioalkalivibrio
nitratireducens and Thioalkalivibrio paradoxus were reported which also reduced
NO 2 and H 2 NOH to NH 4 , as well as SO 2 3 to H 2 S[ 94 , 98 - 100 ]. Both Onr enzymes
showed specific structural features distinguishing them from pentaheme NrfA
enzymes: (i) the covalent Tyr-Cys bond in the active site, (ii) the hexameric
architecture resulting in the formation of a void space inside the hexamer, and
(iii) the product channel that opens into the void interior space of the hexamer.
These structural features might explain the higher nitrite reductase activity and
the greater preference for nitrite than for sulfite as a substrate compared to NrfA
[ 99 , 100 ]. The heme arrangement within each Onr monomer is highly reminiscent
of the octaheme Hao from Nitrosomonas europaea which oxidizes H 2 NOH to NO 2
[ 101 ] but also reduces either nitrite or NO to ammonia in the presence of a strong
reductant [ 102 , 103 ]. Notably, the siroheme-[4Fe-4S]-dependent sulfite reductase
from Mycobacterium tuberculosis also contains a covalent Tyr-Cys bond in the
active site, in close proximity to the siroheme cofactor. Removal of this covalent
bond by site-directed mutagenesis impaired catalytic activity, suggesting that it is
important for the enzymatic reaction [ 104 ].
Taken together, it appears that the multiheme cytochrome classes designated 2.1
to 2.7 in Table 1 share the property of extracytoplasmic ammonia production.

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6 Environmental Issues and Conclusions

As outlined above, ammonia-producing cytochromes c of the MCC family are
abundant enzymes in various habitats on Earth. However, the detection (not to
mention the quantification) of the corresponding genes by PCR-based methods is
hampered by primer design, which generally relies on suitable primary structure
alignments of representative enzymes. In the case of nrfA gene amplification,
it appears that genes encoding NrfA enzymes containing a Cys-X-X-Cys-His at