Ligand migration through the internal hydrophobic cavities in human neuroglobin.
Neuroglobin (Ngb), a member of the globin superfamily, was found in the brain of vertebrates and is suggested to play a neuroprotective function under hypoxic conditions by scavenging nitrogen monoxide (NO) through a dioxygenase activity. In order for such a reaction to efficiently take place and to minimize the release of reactive intermediates in the cytosol, the cosubstrates O(2) and NO and other unstable reaction intermediates should bind sequentially to docking sites in the protein matrix. We have characterized the accessibility of these sites by analyzing the geminate CO rebinding kinetics to the heme moiety observed upon nanosecond flash photolysis of the Ngb-CO complex encapsulated in silica gels. The geminate rebinding phase showed a remarkable complexity, revealing the presence of a system of secondary docking sites where ligands are stored for hundreds of microseconds. Most kinetics steps display little temperature dependence, demonstrating that ligands can easily migrate through the cavities, except for the slowest reaction intermediate, possibly reflecting a structural conformational change reshaping the system of cavities. This conformational change is unrelated with distal His E7 binding to the heme, as it persists for the HE7L mutant. Overall, data are consistent with the presence of a discrete system of docking sites, possibly acting as reservoirs for the putative cosubstrates and for other reactive species involved in the physiologically relevant reaction.
Dipartimento di Fisica, National Enterprise for nanoScience and nanoTechnology CNR-INFM, Università degli Studi di Parma, 43100 Parma, Italy.
Stefania
S
Abbruzzetti
Serena
S
Faggiano
Stefano
S
Bruno
Francesca
F
Spyrakis
Andrea
A
Mozzarelli
Sylvia
S
Dewilde
Luc
L
Moens
Cristiano
C
Viappiani
D002248:Carbon Monoxide / Q000378:metabolism*; D005914:Globins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D000078787:Neuroglobin; D009569:Nitric Oxide / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D058428:Silica Gel; D012822:Silicon Dioxide
D008024:Ligands; D009419:Nerve Tissue Proteins; D000078787:Neuroglobin; D009569:Nitric Oxide; D058428:Silica Gel; D012822:Silicon Dioxide; D002248:Carbon Monoxide; D005914:Globins; D010100:Oxygen