The Production of Ammonia by Multiheme Cytochromes c - The Metal-Driven Biogeochemistry of Gaseous Compounds in the Environment - page 218

¼ 116 mV 3 ½
ð

HSO 3 þ 6e þ 6H þ ! HS þ 3H 2 OE 0 0

HSO 3 = HS

18 Þ

NH 2 OH was converted ten times faster than its O-methyl derivative by the
assimilatory sulfite reductase which brought the authors to the conclusion that the
substrate interacted through its nitrogen moiety with the iron center, as confirmed
later by X-ray crystallography. Notably, ammonia-forming NrfA does also catalyze
reaction ( 18 ) as discussed recently [ 25 ].
Approximately two decades ago, Cole and coworkers located the structural gene
( nrfA ) for cytochrome c 552, the terminal reductase of the formate-dependent
pathway for nitrite reduction to ammonia (nrf, reduction of nitrite by formate) on
the E. coli chromosome [ 50 ]. This enzyme was also called ammonia-forming
cytochrome c nitrite reductase, or cytochrome c nitrite reductase. The corres-
ponding DNA sequence was described to encode a tetraheme c -type cytochrome,
based on the presence of four Cys-X-X-Cys-His heme-binding motifs, in contrast to
the hexaheme cytochrome c reported earlier on the basis of biochemical and
spectroscopic data. Within that sequence, the authors also found a Cys-X-X-Cys-
Lys motif, which they did not assign to a heme-binding site at that time, in
agreement with analytical data (heme and Fe content) for the cytochrome c nitrite
reductase from S. deleyianum and W. succinogenes [ 40 , 50 - 54 ].
NrfA displays typical c -type cytochrome absorption spectra, with absorption
maxima at 280, 409, and 534 nm, and a shoulder at 615 nm in the oxidized
(as isolated) state [ 55 ]. The shoulder at 615 nm can be assigned to a high-spin
Fe(III) heme center [ 40 ]. Upon reduction with sodium dithionite, new maxima
appear at 420, 523, and 553 nm. In the second derivative mode, the spectra reveal
minima at 419, 427, 523, 530, 550, and 555 nm. The maxima at 419 and 550 nm can
be assigned to low-spin Fe(II) heme centers, whereas those at 427 and 555 nm are
consistent with spectral properties of high-spin Fe(II) heme [ 56 , 57 ].
The first two crystal structures were reported for the enzymes from
S. deleyianum [ 58 ] and W. succinogenes [ 59 ] (enzyme class 2.1 in Table 1 ; Figure 3 )
and revealed a homodimeric architecture for NrfA, with five tightly packed c -type
heme centers in each subunit [ 42 ]. Hereafter, more three-dimensional structures
were published, such as for NrfA from E. coli (enzyme class 2.4 in Table 1 )[ 60 ] and
D. desulfuricans ATCC27774 [ 61 ], and for the structure of the NrfA from
D. vulgaris Hildenborough in complex with a second multiheme protein, desig-
nated NrfH.
Note that the cytochrome c nitrite reductases from sulfate-reducing bacteria
D. desulfuricans and D. vulgaris Hildenborough were usually co-purified with
their physiological electron donor, the tetraheme NrfH, as a complex with a well-
defined NrfA:NrfH 2:1 stoichiometry [ 62 ]. This organization was confirmed by the
X-ray structure of the (NrfA 2 H) 2 nitrite reductase complex from D. vulgaris
Hildenborough, which represented a major breakthrough in our understanding of
the dissimilatory reduction of nitrite to ammonia [ 63 ]. Recently, the structure of
S. oneidensis NrfA became available (enzyme class 2.3 in Table 1 ). This enzyme is