Like in other sensor proteins (19
), rebinding of NO and CO with heme was found to be efficient, indicating that the heme pocket acts as an effective ligand trap. Ultrafast recombination of NO and heme is often used to probe the heme environment (17
). For DosS and DosT, picosecond heme-NO rebinding is very efficient and the kinetic properties are very similar, both in the ferrous and ferric state (), although the overall kinetics are somewhat slower in DosS and in the ferric state the escape probability is also higher for DosS, pointing at a higher ligand mobility.
Picosecond CO rebinding in DosS is sizeable but modest (7%) (in many heme proteins, geminate CO rebinding does not occur (17
)), and can be described by a single exponential (280 ps). Here, in the simplest model, upon CO dissociation competition occurs between occupation (in ~300 ps) of a site from which rebinding is very inefficient and rebinding with heme (in ~4 ns). In contrast, in DosT, more extensive and multiexponential heme-CO rebinding is observed, suggesting transiently a heterogeneous distribution of configurations from which rebinding is possible. Investigation of the DosT mutants () indicates that the R87H mutation alters the CO rebinding kinetics towards those observed for DosS. This indicates that the flexibility of Arg87 (His in DosS) in particular allows dissociated CO to occupy a range of conformations in the distal heme cavity, including those from which rebinding to the heme can occur in competition with escape through a ligand entry and exit tunnel, which has been proposed to start out near this residue (15
). In DosS and in the DosT R87H substitution, the rigid His residue may block access of dissociated CO to the heme and/or facilitate access to the ligand tunnel. The opposite effect of the R87A mutation is in agreement with the proposed steric role of this residue at position 87. We remark that the difference in CO binding properties between DosT and DosS must involve the subtle interplay between a number of residues and not uniquely the His residue, as in the R87H/N167T double mutant CO rebinding is more extensive than in the R87H single substitution and the WT DosS protein. Furthermore the effects of the R87H and N167T mutations are not additive.
The most remarkable differences between DosT and DosS that we observed concern O2
dissociation patterns. DosT displays a very low O2
photodissociation yield, similar to what has been observed in the PAS-domain sensor proteins FixL and Ec
), implying that the heme domain acts as an effective O2
trap. By contrast, in DosS the yield is much higher and approaches that of the oxygen storage protein myoglobin (, ). As previously shown for FixL (21
), these differences probably arise from very early dynamic processes occurring within the 100 fs timescale and involving interactions with distal residues that can keep O2
close to the heme after dissociation, thus favoring ultrafast rebinding. In FixL, a hydrogen bond between a distal arginine and O2
plays an important role in this interaction (20
). In DosT, Tyr169, that forms a strong hydrogen bond with the heme-ligated O2
), is likely implicated in such interactions. Indeed, mutation of this tyrosine to phenylalanine, which eliminates this H-bond, results in a much higher O2
escape yield (, ). Our MD simulations indicate that this occurs by releasing the motional restrictions on the 100 fs timescale imposed by the H-bond (). Yet, this finding does not explain the difference between DosT and DosS in this respect, as DosS also carries Tyr at the corresponding position. Mutation of the nearby non-conserved residues Arg87 and Asp167 in DosT did not lead to significantly higher escape yields, indicating that yet to be identified further differences between DosT and DosS are involved. However, the ensemble of our results does strongly suggest that the hydrogen bond between the distal tyrosine and O2
, although clearly present (16
), is weaker in DosS than in DosT. The observed insensitivity of the oxygen binding and autoxidation properties of DosS towards mutation of the distal tyrosine (11
) is in agreement with this proposal. Determination of the crystal structure of the DosS sensor domain oxycomplex may further clarify this issue.
The rate of O2
escape from the protein due to thermal dissociation of the heme Fe-O2
bond (the oxygen off rate) has been suggested to be similar for DosT and DosS, although a directly determined O2
off rate for DosT is not available (7
). If this is correct, and assuming that the intrinsic rate for thermal dissociation of the Fe-O2
bond is similar for DosT and DosS, our data, showing a much higher escape yield for DosS than for DosT on the time scale of 4 ns, suggests that in DosS additional O2
rebinding phases take place in the time window of 4 ns to ~1 ms (that has not been investigated so far). This would imply that in DosS rebinding may take place simultaneously while the ligand switching process is already in progress, a feature that may make fine tuning of the sensor efficiency more sensitive to environmental factors. By contrast, the DosT sensor functions with a mechanism where the switch would fully proceed once the oxygen has escaped the heme pocket (a relatively rare event), as we previously proposed for FixL and Ec
). In this view, the initially operating DosT sensor acts more like other heme-based gas sensors (see Introduction).
The most prominent effect of our mutation studies is observed for the Y169F mutant, both on CO and O2
dynamics. This is not surprising, as Tyr169 is in closest contact with the ligand in the available X-ray structures and is proposed to be involved in the switching pathway (15
). However it is interesting to note that the effect is inverted for CO and O2
: whereas the hydrogen bond with Tyr169 clearly constrains O2
in the heme pocket as discussed above, allowing much more O2
escape from the heme pocket in the mutant, the same Y169F mutation leads to a much lower escape yield for CO. Assuming this latter effect is due to the difference in hydrogen bonding capacity, it may reflect suppression of a hydrogen bond of Tyr169 with CO or, alternatively, a different site. The first possibility is unlikely as in this case hydrogen bonding would be expected to lead to less rather than more CO escape, as for instance observed in the R220H mutation in FixL (20
). Furthermore, at least in DosS, hydrogen bonding of the distal tyrosine to heme-bound CO hardly occurs (16
). Regarding the second possibility, Tyr169 has been invoked to play a key role in the initial signalling pathway. One possible mechanism involves locking the residue in a different configuration in the presence of other ligands than O2
via interaction with another hydrogen-bond partner, either a residue or a water molecule. Such a mechanism has been invoked to explain the ligand discrimination effect of the corresponding tyrosine in DosS (16
). In this mechanism, mutation to Phe would unlock the distal residue so that it can accommodate a position similar to that in the oxygen bound form and favour CO maintenance close to the heme iron after dissociation and thus enhance fast geminate recombination and decrease the escape yield. Our results suggest that such a hydrogen bonding “switch” takes place in DosT signal transmission and also that Tyr169 plays an important role in ligand discrimination, as it does in DosS (16
). Further detailing of the molecular mechanism, and in particular possible hydrogen bond partners in the active form, must await determination of the crystal structure of the CO-bound form or application of appropriate time-resolved vibrational spectroscopic techniques.