Myoglobin Dynamics Measured With Vibrational Echo Experiments

K D Rector,M D Fayer

doi:10.1155/1999/83895

Abstract

Ps infrared vibrational echo experiments on myoglobin and myoglobin mutants are presented. The vibrational dephasing experiments examine the influence of protein dynamics on the CO ligand, at the active site of myoglobin, from low temperature to physiologically relevant temperatures. The vibrational echo results are combined with measurements of the CO vibrational lifetime to yield the homogeneous pure dephasing. The pure dephasing is the Fourier transform of the homogeneous linewidth with the lifetime contribution removed. The mutant H64V protein's CO vibrational pure dephasing rate is ∼20% slower (narrower pure dephasing linewidth) than the native protein at all temperatures, although the only difference between the two proteins is the replacement of the native’s polar distal histidine by a non-polar valine. The mutant H93G(N-MeIm) pure dephasing is identical to the native's, despite the severing of the only covalent bond between the heme and the globin. These results provide insights into the mechanisms of the transmission of protein fluctuations to the CO ligand bound at the active site.

Highlights

This paper presents a study of horseheart whale carbonmonoxy myoglobin (Mb-CO) and a comparison of its dynamics to two myoglobin mutants
The vibrational echo experiments provide a method for the removal of the inhomogeneous broadening from a vibrational spectrum yielding the dynamical information contained in the homogeneous spectrum
We have performed vibrational echo and lifetime relaxation measurements on CO bound to the active site of myoglobin and two myoglobin mutants

Summary

INTRODUCTION

This paper presents a study of horseheart whale carbonmonoxy myoglobin (Mb-CO) and a comparison of its dynamics to two myoglobin mutants. A substantial literature exists that explores the structure and binding kinetics of CO at the active site of Mb using a variety of techniques, including X-ray crystallograpy [3, 4], 13C NMR [5], time dependent optical spectroscopy [6,7,8,9], Raman spectroscopy [10, 11], and mid-infrared absorption spectroscopy [12,13,14] While these methods yield a wealth of information about the equilibrium structure of Mb-CO and the CO binding kinetics, they provide only indirect dynamical information about the protein with a ligand bound to the active site. To more fully understand the nature of protein dynamics communicated to the active site in Mb, we performed a detailed temperature dependent vibrational echo study of a mutant of Mb in which the polar distal histidine is replaced with non-polar valine (H64V). Preliminary results on another mutant, H93G(N-MeIm), are discussed

THE VIBRATIONAL ECHO METHOD AND EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

CONCLUDING REMARKS