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redundancy but that they play distinct roles in sensing changing O
2
tension.
They indicate that DosT appears to first respond to a decline in O
2
tension
when
M. tuberculosis
is gradually transited from aerobic to anaerobic condi-
tions (
Kim et al., 2010
). DosS and DosT show the equilibrium dissociation
constants for O
2
of 3 and 26 mM, respectively (
Sousa et al., 2007
), which is
consistent with the above results.
4.3.2 Crystal structures of the GAF-A domains of DosS and DosT
The crystal structures of the GAF-A domains of DosS (residues 63-210) and
DosT (residues 61-208) have been reported (
Cho, Cho, Kim, Oh, & Kang,
2009; Podust, Ioanoviciu, & Ortiz de Montellano, 2008
). They show a sim-
ilar fold each other, as expected from amino acid sequences homology,
which consist of one five-stranded antiparallel
b
-sheet and four
a
-helices.
A haem is located in the cavity between the
b
-sheet and the loop region
covering the sheet. The haem is tethered to the protein by a histidine
(H149 and H147 for DosS and DosT, respectively) from a long loop con-
necting the
b
3- and
b
4-strands at the proximal position of the haem. The
haem plane is roughly perpendicular to the sheet and shielded by the
a
2
helix and
a
2-
a
3 loop from solvent (
Cho et al., 2009; Podust et al., 2008
).
The crystal structure of DosT reveals that Tyr169 forms a hydrogen bond
with the haem-bound O
2
in DosT (
Podust et al., 2008
). Though the struc-
ture of the oxy form of DosS is not determined, Tyr171 in DosS
(corresponding to Tyr169 in DosT) is thought to form a similar hydrogen
bond with the haem-bound O
2
judging from the resonance Raman studies
combined with site-directed mutagenesis (
Ioanoviciu, Meharenna,
Poulos, & Ortiz de Montellano, 2009; Yukl,
Ioanoviciu, Ortiz de
Montellano, & Mo
¨
nne-Loccoz, 2007
).
4.3.3 Ligand binding to the haem in DosS and DosT
DosS and DosT contain a 5-coordinated, high-spin state of ferrous haem
that binds an exogenous ligand such as O
2
, CO, or NO as an axial ligand
of the haem (
Ioanoviciu, Yukl, Mo¨nne-Loccoz, & Ortiz de Montellano,
2007; Kumar, Toledo, Patel, Lancaster, & Steyn, 2007; Yukl et al., 2008,
2007, 2011
). The resonance Raman spectra of the oxy DosS show the
n
(Fe-O
2
) band at 563 cm
1
that is perturbed by H
2
O/D
2
O exchange,
supporting the presence of a hydrogen bond from a distal residue to the
haem-bound O
2
(
Yukl et al., 2007
). There are two conformers for the
haem-ligand complexes in the NO- and CO-bound forms of DosS. While
one conformer is a major population in which the haem-bound CO or NO
