Ligand binding dynamics to the heme domain of the oxygen sensor Dos from Escherichia coli.
In the heme-based oxygen sensor Dos from Escherichia coli, one of the axial ligands (Met 95) of a six-coordinate heme can be replaced by external ligands such as O(2), NO, and CO, which causes a switch in phosphodiesterase activity. To gain insight into the bidirectional switching mechanism, we have studied the interaction of ligands with the sensor domain DosH by flash photolysis experiments with femtosecond time resolution. The internal ligand can be photodissociated from the ferrous heme and recombines with time constants of 7 and 35 ps. This is somewhat slower than recombination of the external ligands NO, with which picosecond rebinding occurs with unprecedented efficiency (>99%) with a predominant phase of approximately 5 ps, and O(2) (97% in 5 ps, Liebl, U., Bouzhir-Sima, L., Négrerie, M., Martin, J.-L., and Vos, M. H. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 12771-12776). Dissociated CO displays geminate rebinding in 1.5 ns with a very high yield (60%). Together these results indicate that the heme environment provides a very tight pocket for external ligands, presumably preventing frequent switching events. Additional CO dissociation and rebinding experiments on a longer time scale reveal that (a) Met 95 binding, in 100 micros, occurs in competition with bimolecular CO binding, and (b) subsequent replacement of Met 95 by CO on the millisecond time scale occurs faster than in rapid-mixing experiments, suggesting a slow further relaxation. A minimal ligand binding model is proposed that suggests that Met 95 displacement from the heme is facilitated by the presence of an external ligand in the heme environment. Furthermore, the orders of magnitude difference between Met 95 binding after dissociation of internal and external ligands, as well as the spectral characteristics of photodissociation intermediates, indicate substantial rearrangement of the heme environment associated with ligand sensing. Further remarkable observations include evidence for stable (>4 ns) photooxidation of six-coordinate ferrous heme, with a quantum yield of 4-8%.
Liebl
Ursula
U
Laboratory for Optical Biosciences, INSERM U451, CNRS UMR 7645, Ecole Polytechnique-ENSTA, 91128 Palaiseau Cedex, France.
Bouzhir-Sima
Latifa
L
Kiger
Laurent
L
Marden
Michael C
MC
Lambry
Jean-Christophe
JC
Négrerie
Michel
M
Vos
Marten H
MH
D001669:Biochemical Phenomena; D001671:Biochemistry; D002248:Carbon Monoxide / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006418:Heme / Q000737:chemistry*; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008027:Light; D008956:Models, Chemical; D009569:Nitric Oxide / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry; D013997:Time Factors
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D006420:Hemeproteins; D008024:Ligands; C406529:dosH protein, E coli; D009569:Nitric Oxide; D006418:Heme; D002248:Carbon Monoxide; D010100:Oxygen