Vibrational Stark Spectroscopy in Proteins: A Probe and Calibration for Electrostatic Fields

We report the first measurement of the vibrational Stark effect in a protein, providing quantitative information on the sensitivity of a vibrational transition to an applied electric field. This can be used to interpret changes in the vibrational frequency that are often observed when amino acids are changed or when a protein undergoes a structural change in terms of the change in the internal or matrix electric field associated with the perturbation. The vibrational Stark effect has been measured for the vibration of CO bound to the heme iron in myoglobin. The vibrational Stark effect is surprisingly large, giving a Stark tuning rate of (2.4/f) cm-1/(MV/cm), where f is the local field correction; this is nearly 4 times larger than for free CO. It is also found that the change in dipole moment is parallel to the transition moment; that is, the change in dipole moment is in the direction perpendicular to the heme plane. Vibrational Stark effect data are also reported as a function of pH, for various mutants, for a modified picket fence porphyrin, and for cytochrome c. The Stark tuning rate is found to be very similar in all cases, indicating that the CO stretch frequency for CO bound to the heme iron is a sensitive and anisotropic local detector of changes in the electrostatic field. This information is used to evaluate electrostatics calculations for heme proteins. Electrostatic interactions are central to understanding the properties of molecules in the condensed phase and ar