Structural Dynamics of Myoglobin:  Effect of Internal Cavities on Ligand Migration and Binding

Abstract

Using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures, we have studied CO binding to the heme and CO migration among cavities in the interior of sperm whale carbonmonoxy myoglobin (MbCO) after photodissociation. Photoproduct intermediates, characterized by CO in different locations, were selectively enhanced by laser illumination at specific temperatures. Measurements were performed on the wild-type protein and a series of mutants (L104W, I107W, I28F, and I28W) in which bulky amino acid side chains were introduced to block passageways between cavities or to fill these sites. Binding of xenon was also employed as an alternative means of filling cavities. In all samples, photolyzed CO ligands were observed to initially bind at primary docking site B in the vicinity of the heme iron, from where they migrate to the secondary docking sites, the Xe4 and/or Xe1 cavities. To examine the relevance of these internal docking sites for physiological ligand binding, we have performed room-temperature flash photolysis on the entire set of proteins in the CO- and O(2)-bound form. Together with the cryospectroscopic results, these data provide a clear picture of the role of the internal sites for ligand escape from and binding to myoglobin.

Ligand Migration and Binding in the Dimeric Hemoglobin ofScapharca inaequivalvis,

“…In contrast, the Xe1 site in Mb can be filled with Xe to 97% under similar conditions (66). However, a bulky indole moiety at position G5 in L104W MbCO completely occludes this cavity (28). Moreover, in HbI, the accessibility of the HbI Xe1 site depends on the ligation state of the protein.…”

“…(FTIR) cryospectroscopy to study the migration of carbon monoxide (CO) among cavities within the protein interior, using the CO molecule as a sensitive probe of the local environment (27)(28)(29)(30)(31). At physiological temperature, these cavities serve as transient ligand docking sites so that the ligand shuttles back and forth among the docking sites within the protein after dissociation from the heme iron.…”

“…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.…”

“…FTIR transmission spectra were collected between 1800 and 2400 cm -1 at a resolution of 2 cm -1 using a FTIR spectrometer equipped with an InSb detector (IFS 66v/S, Bruker, Karlsruhe, Germany). Sample loading and cryospectroscopy equipment have been described previously (28)(29)(30)58 Temperature derivative spectroscopy (TDS) is a two-dimensional spectroscopic method that employs temperature as a second dimension for studying thermally activated rate processes governed by enthalpy barrier distributions (59-61). These distributions are universally observed in ligand binding to heme and other proteins at cryogenic temperatures and result from conformational heterogeneity (6,62).…”

“…Therefore, we have chosen to investigate CO ligand dynamics within HbI with a similar strategy as for Mb, namely by combining x-ray crystallography and FTIR cryospectroscopy (27)(28)(29)(30). A series of mutants was prepared to modify protein-ligand interactions, based on the experience gained from earlier studies on Mb (28).…”