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Probing Electric Fields in Protein Cavities by Using the Vibrational Stark Effect of Carbon Monoxide

Hartwig Lehle ^*, Jan M. Kriegl ^*, Karin Nienhaus ^*, Pengchi Deng ^*, Stephanus Fengler ^* and G. Ulrich Nienhaus ^* 

^* Department of Biophysics, University of Ulm, D-89081 Ulm, Germany; and Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 USA

Correspondence: Address reprint requests to Gerd Ulrich Nienhaus, E-mail: uli{at}uiuc.edu.

To determine the magnitude and direction of the internal electric field in the Xe4 cavity of myoglobin mutant L29W-S108L, we have studied the vibrational Stark effect of carbon monoxide (CO) using infrared spectroscopy at cryogenic temperatures. CO was photodissociated from the heme iron and deposited selectively in Xe4. Its infrared spectrum exhibits Stark splitting into two bands associated with CO in opposite orientations. Two different photoproduct states can be distinguished, C' and C'', with markedly different properties. For C', characteristic temperature-dependent changes of the area, shift, and width were analyzed, based on a dynamic model in which the CO performs fast librations within a double-well model potential. For the barrier between the wells, a height of 1.8 kJ/mol was obtained, in which the CO performs oscillations at an angular frequency of 25 cm^–1. The magnitude of the electric field in the C' conformation was determined as 11.1 MV/cm; it is tilted by an angle of 29° to the symmetry axis of the potential. Above 140 K, a protein relaxation leads to a significantly altered photoproduct, C'', with a smaller Stark splitting and a more confining potential (barrier >4 kJ/mol) governing the CO librations.

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