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to the Fe atom (
Howes et al., 2011
). The ferrous state shows a mixture of a
predominant hexacoordinated low-spin state (Soret band at 421 nm and
Q bands at 528 and 559 nm) and a pentacoordinated high-spin state (shoul-
der at 440 nm) (
Fig. 8.6
B;
Giordano et al., 2011
). These spectra are in mar-
ked contrast to those of monomeric Mb, in which the Fe atom is
pentacoordinated. In fact, the deoxygenated ferrous form has a broad peak
at 556 nm (
Fig. 8.6
B), whereas the ferric form exhibits two peaks at 504 and
632 nm (
Fig. 8.6
A;
Antonini & Brunori, 1971
).
Based on the spectroscopic data and molecular-dynamics simulation
(
Howes et al., 2011
), it has been shown that either TyrCD1 or TyrB10
can coordinate the ferrous atom. Although His is the most common residue
that coordinates the Fe atom, Tyr coordinates Fe of ferrous
Herbaspirillum
seropedicae
Hb (
Razzera et al., 2008
) and of ferrous and ferric
Chlamydomonas
Hb (
Couture et al., 1999; Das et al., 1999; Milani et al., 2005
).
6.2.3 Reactivity
Reversible hexa- to pentacoordination of the haem-Fe atom modulates the
reactivity of
Ph
-2/2HbO; in fact, the cleavage of the haem distal
Fe-TyrCD1 or Fe-TyrB10 bonds is the rate-limiting step for the association
of exogenous ligands (e.g. O
2
, CO and NO) and (pseudo)enzymatic activ-
ities (
Russo et al., 2013
).
CO binding to
Ph
-2/2HbO displays a rapid spectroscopic phase inde-
pendent of CO concentration, followed by standard bimolecular recombi-
nation. CO-rebinding kinetics show an unusually slow geminate phase,
which becomes dominant at low temperature. While geminate recombina-
tion usually occurs on the ns timescale,
Ph
-2/2HbO displays a component of
about 1
m
s that accounts for half of the geminate phase at 8
C, indicative of a
relatively slow internal ligand binding (
Russo et al., 2013
).
After ligand escape, bimolecular recombination takes place. Second-
order rebinding indicates two major conformations at 25
C, characterised
by CO-association rates that differ by a factor of 20, with pH-dependent
relative fractions. A dynamic equilibriumwas found between a predominant
hexacoordinated low-spin state and a pentacoordinated high-spin state.
A shift in the equilibrium between the two conformations may also provide
a large change in the ligand affinity. The second-order rate constant of the
fast phase (
Russo et al., 2013
) is of the order of 10
7
M
1
s
1
and closely sim-
ilar to that of human Ngb (
Uzan et al., 2004
), whereas the second-order rate
constant of the slow process (
Russo et al., 2013
) is compatible with that of
Mb (
Table 8.4
), being in the range of 10
5
M
1
s
1
(
Springer, Sligar,
