Energy transformations early in the bacteriorhodopsin photocycle revealed by DNP-enhanced solid-state NMR

*Department of Chemistry, Brandeis University, Waltham, MA 02453; and
^‡Department of Chemistry,
^†Francis Bitter Magnet Laboratory, and
^¶Plasma Science and Fusion Center, Massachusetts Institute of Technology, Cambridge, MA 02139

Edited by Ann E. McDermott, Columbia University, New York, NY, and approved December 7, 2007 (received for review July 4, 2007)

Abstract

By exploiting dynamic nuclear polarization (DNP) at 90 K, we observe the first NMR spectrum of the K intermediate in the ion-motive photocycle of bacteriorhodopsin. The intermediate is identified by its reversion to the resting state of the protein in red light and by its thermal decay to the L intermediate. The ¹⁵N chemical shift of the Schiff base in K indicates that contact has been lost with its counterion. Under these circumstances, the visible absorption of K is expected to be more red-shifted than is observed and this suggests torsion around single bonds of the retinylidene chromophore. This is in contrast to the development of a strong counterion interaction and double bond torsion in L. Thus, photon energy is stored in electrostatic modes in K and is transferred to torsional modes in L. This transfer is facilitated by the reduction in bond alternation that occurs with the initial loss of the counterion interaction, and is driven by the attraction of the Schiff base to a new counterion. Nevertheless, the process appears to be difficult, as judged by the multiple L substates, with weaker counterion interactions, that are trapped at lower temperatures. The double-bond torsion ultimately developed in the first half of the photocycle is probably responsible for enforcing vectoriality in the pump by causing a decisive switch in the connectivity of the active site once the Schiff base and its counterion are neutralized by proton transfer.

Footnotes

**To whom correspondence should be addressed. E-mail: herzfeld{at}brandeis.edu
Author contributions: R.G.G. and J.H. designed research; M.L.M.-J., V.S.B., M.K.H., and M.B. performed research; M.L.M.-J., V.S.B., R.G.G., and J.H. analyzed data; and M.L.M.-J., V.S.B., R.G.G., and J.H. wrote the paper.
↵ ^§Present address: Department of Chemistry, University of California, Berkeley, CA 94720.
↵ ^‖Present address: Beam Physics Branch, Plasma Physics Division, Naval Research Laboratory, Washington, DC 20375.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.