Vibrational Stark Spectroscopy of NO Bound to Heme: Effects of Protein Electrostatic Fields on the NO Stretch Frequency

Abstract

The vibrational Stark effect measures the effect of an external electric field on the vibrational (IR) spectrum of a molecule. This technique gives quantitative information on the sensitivity of a vibrational peak position to an electric field. This calibration can be used to evaluate shifts in the vibrational frequency caused by changes in the local electric field in the organized electrostatic matrix of a protein, for example, by mutating amino acid residues near the vibration whose frequency is probed. We report vibrational Stark effect measurements for NO bound to several distal pocket mutants of myoglobin, (Val68Asp, Val68Asn, Val68Glu, and His64Val). These mutations were designed to perturb the electrostatic field near the NO bound to the heme iron. The magnitude of the change in dipole moment, |Δμ|, for the vibration of NO bound to heme is found to be approximately 0.12 D/f, that is, the Stark tuning rate is 2.0/f cm-1/(MV/cm) (where f is the local field correction) for a series of distal pocket mu...