Extended subnanosecond structural dynamics of myoglobin revealed by Laue crystallography

Abstract

Work carried out over the last 30 years unveiled the role of structural dynamics in controlling protein function. Cavity networks modulate structural dynamics trajectories and are functionally relevant; in globins they have been assigned a role in ligand migration and docking. These findings raised renewed interest for time-resolved structural investigations of myoglobin (Mb), a simple heme protein displaying a photosensitive iron-ligand bond. Photodissociation of MbCO generates a nonequilibrium population of protein structures relaxing over a time range extending from picoseconds to milliseconds. This process triggers ligand migration to matrix cavities with clear-cut effects on the rate and yield of geminate rebinding. Here, we report subnanosecond time-resolved Laue diffraction data on the triple mutant YQR-Mb [Leu-29(B10)Tyr, His-64(E7)Gln, Thr-67(E10)Arg] that depict the sequence of structural events associated with heme and protein relaxation from 100 ps to 316 ns and above. The photodissociated ligand rapidly (<0.1 ns) populates the Xe-binding cavity distal to the heme. Moreover, the heme relaxation toward the deoxy configuration is heterogeneous, with a slower phase ( approximately ns) evident in these experiments. Damping of the heme response appears to result from a strain exerted by the E-helix via the CD-turn; Phe-43(CD1), in close contact with heme, opposes tilt until the strain is relieved. A comparison with crystallographic data on wild-type Mb and mutants Leu(29)Phe or Leu(29)Trp suggests that the internal structure controls the rate and amplitude of the relaxation events. A correlation between structural dynamics as unveiled by Laue crystallography and functional properties of Mb is presented.

Fig. 1.

Structural changes in the heme vicinity of YQR-Mb detected at 0.1–316 ns after CO photolysis; the time frames at 316 ps and 1 ns are not shown. (F_light − F_dark) difference electron density maps (red: negative; green: positive; contoured at 3.0 σ, where σ is the standard deviation of the electron density difference) are overlaid on models of YQR-MbCO (yellow, Protein Data Bank code 1MYZ) and YQR-Mb (blue, Protein Data Bank code 1MZ0). For clarity, a magenta bar indicates the position of CO in both the Xe1 and Xe4 pockets.

Fig. 2.

Time dependence of difference electron densities for key structural features. The numerical values reflect the integral of the positive electron density beyond 3.0 σ and are corrected for variations in photolysis yield. They are normalized so that the negative bound-CO feature is assigned a value of 1. Average values weighted by photolysis yield over the four independent data sets are shown. Error bars correspond to twice the weighted standard deviations between data sets. (a) Key features that appear promptly. (b) Residues involved in the strain of the CD turn, lagging behind. (c) Population of CO in the Xe1 and Xe4 sites and changes in secondary structures; in the latter, the average integrated density per residue is shown.

Fig. 3.

A schematic view of a possible mechanism to account for the structural constraints involved in damping the heme response after CO photolysis (see text). Displacements caused by the transition from the CO-bound state (dark gray) to the deoxy state (light gray), together with relevant residues and the heme, are shown. Arrows highlight stereochemical clashes.