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COMMENTS Comment on “Ultrafast Dynamics of Myoglobin without the Distal Histidine: Stimulated Vibrational Echo Experiments and Molecular Dynamics Simulations”1,† Anne Goj and Roger F. Loring* Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell UniVersity, Ithaca, New York 14853 ReceiVed: June 18, 2007; In Final Form: August 17, 2007 Kinetics and thermodynamics of ligand binding in myoglobin are strongly influenced by the interaction between the ligand and the distal histidine (H64), the most polar residue close to the binding site.2 The conformation of this residue is also implicated in the origin of the spectroscopic substates associated with the multiple peaks in the infrared absorption spectrum of bound CO in carbon monoxymyoglobin (MbCO).2,3 The mutant myoglobin H64V has the nonpolar residue valine substituted for the distal histidine4 so that spectroscopic studies of H64VCO can reveal protein dynamics that are present in the wildtype species but may be obscured by the interaction between H64 and CO. Finkelstein, et al.1 have compared spectrally resolved infrared stimulated echoes of the CO vibration in H64V-CO to previous vibrational echo studies of wild-type MbCO.3 In aqueous solution at room temperature, the vibrational dephasing rate is decreased by ∼50% in the mutant. Measured vibrational echoes in H64V were also compared1 to calculations3 in which the vibrational dephasing of CO is determined by electric field fluctuations at the active site, computed from molecular dynamics simulations of H64V-CO in water using the MOIL force field.5 The analogous procedure applied to MbCO yielded results in quantitative agreement with measured echoes.3 Although calculated echoes for H64V-CO decay more slowly than calculated echoes for MbCO, the calculated echoes for H64V-CO consistently decay more rapidly1 than the measured echoes for H64V-CO. Here, we present an interpretation for the finding that these calculations yield better agreement with experiment for MbCO than for H64V-CO. Figure 12 in ref 3 shows that electric field fluctuations at the active site of MbCO generated by the solvent are anticorrelated with electric field fluctuations from the protein, leading to substantial cancellation in the effect of solvent and protein motions on the CO vibration. Since the publication of ref 1, we have shown for H64V-CO that the solvent electric field fluctuations that are anticorrelated with those from the protein arise primarily from water molecules in the first hydration shell about the protein exterior.6 This cancellation leaves an influence on CO vibrational dephasing from motions of water molecules beyond the first hydration layer, whose dynamics7 are closer to those of bulk water. The MOIL force field5 employs the TIP3P water model, which predicts both rotational and translational dynamics in bulk water that are too fast,8 suggesting the possibility that these too facile solvent dynamics are the cause † Dr. Michael Fayer, the corresponding author of the original paper, is in agreement with this Comment.
Figure 1. Measured and calculated vibrational echo decays at Tw ) 0.5 ps and linear spectra (inset) for myoglobin mutant H64V-CO. Dotted curves show measurements,1,9 solid curves are calculated with the TIP3P water model,1 and dashed curves show calculations with the SPC/E water model.
for the too rapid echo decays predicted for H64V-CO. The TIP3P water model was also employed in our calculations for MbCO,3 but because of the strong interactions between H64 and CO, the water contribution to dephasing was relatively smaller. We have tested this hypothesis by repeating the H64V-CO calculations using the SPC/E water model, which predicts slower and more accurate8 molecular motions in bulk water than does TIP3P. Figure 1, with a format that is similar to Figure 5 of ref 1, shows the measured1 (dotted line) three-pulse echo as a function of τ, the delay time between the first two pulses, with the time between the second and third pulses set to Tw ) 0.5 ps. Calculated echoes are shown by the solid line (TIP3P) and by the dashed line (SPC/E). Echo calculations assume impulsive excitation. Corresponding measured9 and calculated infrared absorption line shapes of H64V-CO are shown in the inset. We have calculated echoes and line shapes from equilibrium autocorrelation functions of electric field fluctuations at the active site using the same approximations and procedures described in ref 1, without adjustable fitting parameters. The vibrational echo with SPC/E water is significantly slower and, thus, closer to the measured result than the echo calculated with TIP3P water. The largely inhomogeneously broadened linear spectrum is less sensitive to the change in water model than is the vibrational echo. Echo calculations not shown here using the SPC water model,8 which like TIP3P overestimates relaxation rates in bulk water,8 were nearly identical to the results with TIP3P. Since the contributions to the fluctuating electric field at the active site from different regions of the protein and solvent may be positively or negatively correlated,3 it is difficult to identify the microscopic origin of the remaining discrepancy in Figure 1 between the echo calculated with SPC/E water and the measured result. However, these calculations demonstrate that a more accurate representation of water dynamics brings the echo calculation into better agreement with the experimental data. Figure 1 illustrates a procedure in which comparison of measured and calculated nonlinear spectroscopic observables can test the predictions by empirical force fields of fast
10.1021/jp074711w CCC: $37.00 © 2007 American Chemical Society Published on Web 10/16/2007
Comments molecular motions. These particular results also emphasize the influence of solvent dynamics7 on the nonlinear infrared spectroscopy of proteins. Acknowledgment. We thank I. J. Finkelstein and M. D. Fayer for discussions and S. Kim and M. D. Fayer for the unpublished absorption spectrum in Figure 1. This material is based upon work supported by the National Science Foundation through Grant No. CHE0413992. References and Notes (1) Finkelstein, I. J.; Goj, A.; McClain, B. L.; Massari, A. M.; Merchant, K. A.; Loring, R. F.; Fayer, M. D. J. Phys. Chem. B 2005, 109, 16959.
J. Phys. Chem. B, Vol. 111, No. 44, 2007 12939 (2) Phillips, G. N.; Teodoro, M. L.; Li, T.; Smith, B.; Olson, J. S. J. Phys. Chem. B. 1999, 103, 8817. (3) Merchant, K. A.; Noid, W. G.; Akiyama, R.; Finkelstein, I. J.; Goun, A.; McClain, B. L.; Loring, R. F.; Fayer, M. D. J. Am. Chem. Soc. 2003, 125, 13804. (4) Quillin, M. L.; Arduini, R. M.; Phillips, G. N. J. Mol. Biol. 1993, 234, 144. (5) Elber, R.; Roitberg, A.; Simmerling, C.; Goldstein, R.; Li, H.; Verkhivker, G.; Keasar, C.; Zhang, J.; Ulitsky, A. Comput. Phys. Commun. 1994, 91, 159. (6) Goj, A. Vibrational Dephasing in a Mutant Myoglobin; Ph.D. Dissertation, Cornell University, 2007. (7) Goj, A.; Loring, R. F. Chem. Phys. 2007, in press. (8) van der Spoel, D.; van Maaren, P. J.; Berendsen, J. C. J. Chem. Phys. 1998, 108, 10220. (9) Kim, S.; Fayer, M. D. personal communication, 2007.
