Dynamics of Myoglobin−CO with the Proximal Histidine Removed:  Vibrational Echo Experiments

Abstract

Picosecond infrared vibrational echo measurements from 60 to 300 K on CO bound to the active site of a mutant myoglobin, H93G(N-MeIm), are presented and compared to measurements on native myoglobin and on the mutant H64V. Although in H93G(N-MeIm) (the proximal histidine replaced by glycine, with exogenous N-methylimidazole in the proximal histidine pocket, covalently bound to Fe), the only covalent linkage between heme−CO and the protein is broken, there is no change in the temperature-dependent vibrational pure dephasing time, T 2*. The results demonstrate that severing the only covalent bond between the heme and the globin has little or no effect on the protein dynamics detected by vibration of the CO at the active site of myoglobin.

Vibrational Dephasing of Carbonmonoxy Myoglobin

“…Because vibrational echo experiments [5][6][7][8][9][10] probe only the A 1 state of the CO vibrational band, trajectories in which the distal histidine assumes a conformation associated with the A 0 state 25 were discontinued once this event occurred. Configurations were deemed to correspond to the A 0 band if the distance of the H atom bonded to N δ on the distal histidine exceeded a distance of 6.5 Å from the midpoint of the CO bond.…”

“…A novel class of nonlinear vibrational spectroscopic techniques, [1][2][3][4] which are analogs of multidimensional magnetic resonance spectroscopy, can provide a direct measure of such protein structural fluctuations. These measurements include the two-pulse and three-pulse infrared vibrational echoes, which have been applied to ligands bound to heme proteins by Fayer and co-workers [5][6][7][8][9][10] and by Hochstrasser and co-workers. [11][12][13][14] The two-pulse vibrational echo, like its optical and magnetic resonance predecessors, has the capability to extract a homogeneous dephasing time, T 2 , for a vibrational transition that is inhomogeneously broadened.…”

Dynamics of Proteins Encapsulated in Silica Sol−Gel Glasses Studied with IR Vibrational Echo Spectroscopy

“…The time dependent Stark effect causes the CO vibrational frequency to fluctuate, producing dynamic dephasing. 61,66,[73][74][75][76] There is also a contribution to the vibrational echo observable from CO vibrational population relaxation. In carbonmonoxy heme proteins, such as MbCO and HbCO, the vibrational lifetime of the bCO stretch is sufficiently long that the vibrational echo decay is primarily a measure of CO dephasing caused by protein structural fluctuations.…”

Cytochrome c552Mutants: Structure and Dynamics at the Active Site Probed by Multidimensional NMR and Vibration Echo Spectroscopy

“…13,[25][26][27][28][29][30][31][32][33] Multidimensional IR spectroscopy has improved upon the dynamic range of NMR techniques, revealing biochemical dynamics that occur on the picosecond to femtosecond time scales. [34][35][36][37][38][39][40][41] Vibrational echo spectroscopy is a multidimensional IR technique that is sensitive to the relationship between structure and dynamics in heme proteins. [34][35][36][37][38][39][40][41][42][43] These measurements probe the dephasing of a heme-bound CO vibration caused by structural fluctuation of the protein.…”

“…[34][35][36][37][38][39][40][41] Vibrational echo spectroscopy is a multidimensional IR technique that is sensitive to the relationship between structure and dynamics in heme proteins. [34][35][36][37][38][39][40][41][42][43] These measurements probe the dephasing of a heme-bound CO vibration caused by structural fluctuation of the protein. There is also a contribution to the vibrational echo observable from vibrational energy relaxation from the CO vibration to other modes.…”