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the EPR signals of
g
¼
2.52, 2.28, and 1.88, which are similar to those of a
thiolate-bound, low-spin haem such as Rr-CooA (
Aono et al., 1998
).
The MCD spectrum of ferric RcoM2 shows an intense temperature-
dependent, derivative-shaped C term in the Soret region with a crossover
at 419 nm, and another temperature-dependent C term is also present in
the Q-bands region with a crossover at 570 nm (
Marvin et al., 2008
). Fer-
rous RcoM2 shows a temperature-independent A term in the Q-bands
region with a crossover at 560 nm and much less intense temperature-
independent feature in the Soret region with a crossover at 421 nm in its
MCD spectrum (
Marvin et al., 2008
).
In the resonance Raman spectra, the
n
2,
n
3,
n
4, and
n
10 bands are
observed at 1579, 1500, 1371, and 1634 cm
1
; 1580, 1490, 1359, and
1580 cm
1
; and 1575, 1494, 1368, and 1630 cm
1
, for ferric, ferrous,
and CO-bound RcoM2, respectively (
Marvin et al., 2008
). The correlation
between
n
(Fe-CO) (485 cm
1
) and
n
(C-O) (1965 cm
1
) bands indicates
that the proximal ligand
trans
to CO is a neutral His in CO-bound RcoM2.
While the C94S variant of RcoM2 shows the identical spectroscopic fea-
tures to the wild type in the ferrous and CO-bound forms, the ferric RcoM2
shows different UV-vis, EPR, and resonance Raman spectra from those of
the wild type. Though the wild-type RcoM shows the
d
-band at 354 nm
typical for a thiolate-bound haem, a clear
d
-band is not observed in the
C94S variant. The C94S variant shows an axial signals of
g
5.82 and
2.00 instead of a set of rhombic signals observed in the wild-type RcoM2
(
Smith et al., 2012
). Resonance Raman spectrum reveals that the ferric
haem in the C94S variant is a mixture of a low-spin and high-spin states,
where the two bands are observed for both
n
2 and
n
3 at 1562 cm
1
(high
spin)/1577 cm
1
¼
(low spin) and 1493 cm
1
(high spin)/1503 cm
1
(low
spin), respectively.
Based on these spectral features, the coordination structures of the haem
in RcoM2 are proposed as shown in
Fig. 7.1
(
Smith et al., 2012
). RcoM2
shows a ligand exchange upon the change in the oxidation state of the haem
and upon binding CO. Though His74 is retained as the proximal ligand in
the ferric, ferrous, and CO-bound haems, the distal ligand exchange takes
place between the ferric and ferrous haems and between the ferrous and
CO-bound haems. Cys94 coordinated to the ferric haem is replaced by
Met104 upon reduction of the haem to form a 6-coordinate ferrous haem
with His74 and Met104 as the axial ligands. Met104 is replaced by CO upon
reaction of ferrous RcoM2 with CO to form the CO-bound RcoM2.
Amino acid sequence alignments suggest that Met104 is located in the
