Vibrational stark spectroscopy in proteins: A probe and calibration for electrostatic fields

We report the first measurement of the vibrational Stark effect in a protei n, providing quantitative information on the sensitivity of a vibrational t ransition to an applied electric field. This can be used to interpret chang es in the vibrational frequency that are often observed when amino acids ar e changed or when a protein undergoes a structural change in terms of the c hange in the internal or matrix electric field associated with the perturba tion. The vibrational Stark effect has been measured for the vibration of C O bound to the heme iron in myoglobin. The vibrational Stark effect is surp risingly large, giving a Stark tuning rate of (2.4/f) cm(-1)(MV/cm), where fis 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 t ransition moment; that is, the change in dipole moment is in the direction perpendicular to the heme plane. Vibrational Stark effect data are also rep orted 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 boun d to the heme iron is a sensitive and anisotropic local detector of changes in the electrostatic field. This information is used to evaluate electrost atics calculations for heme proteins.