The role of cavities in protein dynamics: Crystal structure of a photolytic intermediate of a mutant myoglobin

Abstract

(1) to account for the efficient fluorescence quenching of internal tryptophans by dioxygen. In the crystal structure of enzymes processing large substrates, a channel involved in the capture and optimal presentation to the catalytic site is seen (2). Cavities and connecting channels identified in the interior space of a protein (3) confer flexibility and alternative packing arrangements that allow rapid transitions between structurally distinct states; in small globular proteins, however, it is not known whether these packing defects are important in controlling function by defining alternative pathways and hosting stations in the diffusion of a ligand to the active site. The ultra-low temperature x-ray diffraction experiment on the sperm whale myoglobin (Mb) mutant presented in this paper supports the viewpoint that pre-existing internal cavities play a major role in controlling the dynamics of ligand binding and can be modified by protein engineering.The role of protein relaxation in Mb has been extensively investigated by laser photolysis because the photosensitivity of the complex of ferrous Mb with CO, O 2 , and NO makes it possible to populate intermediate states and to follow the structural dynamics involved in the relaxation of the protein and the migration of the ligand through the matrix. These diatomic molecules are small enough to migrate rapidly away from the metal and large enough to probe accessibility of atomic-sized cavities that are sparse in the structure and interconnected through fluctuating channels (3-5). The high photosensitivity of the MbCO adduct, with unitary quantum yield (6), makes this derivative an ideal model for time-resolved crystallography (7) using intense synchrotron x-ray sources.Previous crystallographic studies on the photolyzed state of wild-type (wt) sperm whale Mb showed the photodissociated CO* to reside in the heme pocket near the metal but at nonbonding distance, both at room temperature in the first time-resolved nsec diffraction experiment (7) and at ultra-low temperatures (8-10). Differences in the location of CO* observed in the cryogenic x-ray diffraction experiments were attributed (partially or totally) to a variable degree of photolysis related to the experimental protocol and͞or the temperature. The results indicate (see ref. 10 for a discussion) that the photolyzed CO* is located largely 3.6-3.7 Å from the Fe (which moves out of the heme plane by Ϸ 0.3 Å), in agreement with optical absorption and polarized IR time-resolved studies, as well as molecular dynamics calculations (11)(12)(13)(14). A study of the structural dynamics of Mb by x-ray diffraction may provide an answer to some interesting questions, such as: (i) How far could the niche hosting the photolyzed CO* be considered a docking site? (ii) To what extent could this niche be controlled by the nature of the side chains in the distal heme pocket? (iii) Could protein engineering allow one to modify the pathways for ligand migration through cavities and channels within the protein matrix, to and fr...

Ligand binding and protein dynamics in neuroglobin

“…1). Apparently, the distal heme pocket is spacious and allows the ligand to migrate among different docking sites, as has been observed earlier in Mb mutants (20)(21)(22)(23).…”

Ligand Migration and Binding in the Dimeric Hemoglobin ofScapharca inaequivalvis,

“…In the A 1 and A 3 substates, the imidazole side chain is neutral and resides inside the distal pocket, whereas it is doubly protonated and pointed towards the solvent in the A 0 substate (67,68). After photodissociation, the CO ligand transiently occupies specific locations inside Mb, the primary docking site B on top of heme pyrrole C (14-16) and secondary sites C and D that coincide with two of four hydrophobic cavities in the protein interior, labeled Xe4 and Xe1 for their ability to bind Xe (17)(18)(19)69). In each of these sites, the CO ligand gives rise to specific photoproduct spectra, with the stretching frequency fine-tuned by the vibrational Stark effect, the interaction of the CO infrared transition dipole with the electric field at the site (31,63,70,71).…”

“…Sophisticated biophysical experiments, notably x-ray structure analysis (14)(15)(16)(17)(18)(19)(20), optical and infrared spectroscopy (21)(22)(23)(24)(25)(26)(27)(28)(29) applied over wide ranges in time and temperature and on a large number of genetically modified Mbs have led to a detailed picture of ligand binding in this protein. In our laboratory, we have focused on Fourier transform infrared (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).…”

Multiple strategies for O2 transport: from simplicity to complexity

“…Site-directed mutagenesis has confirmed the gating role of HisE7 (45) and has allowed to postulate a role for the system of cavities where ligands can dock temporarily (62). Time-resolved crystallography (63 -65) and cryo-trapping experiments (66,67) showed how the internal Mb cavity system modulates heme reactivity in a subtle interplay with tertiary and side-chain transitions centered on the topological position E7 (68).…”