Ligand binding and conformational motions in myoglobin

Abstract

Myoglobin, a small globular haem protein that binds gaseous ligands such as O2, CO and NO reversibly at the haem iron, serves as a model for studying structural and dynamic aspects of protein reactions. Time-resolved spectroscopic measurements after photodissociation of the ligand revealed a complex ligand-binding reaction with multiple kinetic intermediates, resulting from protein relaxation and movements of the ligand within the protein. To observe the structural changes induced by ligand dissociation, we have carried out X-ray crystallographic investigations of carbon monoxy-myoglobin (MbCO mutant L29W) crystals illuminated below and above 180 K, complemented by time-resolved infrared spectroscopy of CO rebinding. Here we show that below 180 K photodissociated ligands migrate to specific sites within an internal cavity--the distal haem pocket--of an essentially immobilized, frozen protein, from where they subsequently rebind by thermally activated barrier crossing. Upon photodissociation above 180 K, ligands escape from the distal pocket, aided by protein fluctuations that transiently open exit channels. We recover most of the ligands in a cavity on the opposite side of the haem group.

Ligand Migration and Binding in the Dimeric Hemoglobin ofScapharca inaequivalvis,

“…Ligand trapping in this site is especially efficient in HbI mutant M37F (Figure 6) although Phe-37 partially constricts the pathway to the cavity in the A subunit (but not in the B subunit). The same effect has been observed in Mb mutants with aromatic amino acids at position B10, including L29F (93), triple mutant YQR (L29F-H64Q-T67R) (58,80) and especially L29W, for which the Xe4 cavity is a highly efficient ligand trap at cryogenic temperatures (18,29). The Xe4 site is separated from the distal pocket by a narrow channel bordered by Leu-36 (B9), Leu-73 (E11), and Ile-114 (G8), which in wild-type HbI is relatively free from obstruction.…”

“…For example, an exceptionally large internal volume of ∼300 Å 3 exists in neuroglobin, which allows the entire heme prosthetic group to slide deeper into the protein so as to resolve steric conflicts between the exogenous ligand and the distal histidine (97). In the (18,20,28,30,69,93,98). Covalent bond formation to the heme iron can only occur from the primary docking site, and these additional docking sites lower the rebinding probability until the dissociated ligand can finally leave the protein with the aid of a rare protein fluctuation that opens an escape pathway.…”

The dynamic transition in proteins may have a simple explanation

“…[14][15][16][17][18][19][20] The processes for which these associations have generally been made, such as electron transport or ligand binding, [14][15][16] are those involving relatively fast reactions (and under conditions of relatively low hydration, rather than in solution). This is presumably because of a dependence upon the faster motions, which become too slow to allow normal function.…”

Control of Stereoselectivity in an Enzymatic Reaction by Backdoor Access

“…[8][9][10] They have been implicated in controlling substrate entrance, product egress, removal of bound water, cofactor binding, and proton shuttling. [11,12] The stereochemical consequences of having multiple-access pathways to one stereoselective catalytic center, however, have not been investigated so far.…”