Subpicosecond Protein Backbone Changes Detected during the Green-Absorbing Proteorhodopsin Primary Photoreaction Jason J. Amsden, †,⊥ Joel M. Kralj, †,⊥ Logan R. Chieffo, ‡,⊥ Xihua Wang, †,⊥ Shyamsunder Erramilli, †,§,⊥ Elena N. Spudich, | John L. Spudich, | Lawrence D. Ziegler, ‡,⊥ and Kenneth J. Rothschild* ,†,⊥,3 Department of Physics, Boston UniVersity, Boston, Massachusetts 02215, Department of Chemistry, Boston UniVersity, Boston, Massachusetts 02215, Department of Biomedical Engineering, Boston UniVersity, Boston, Massachusetts 02215, Center for Membrane Biology, Department of Biochemistry and Molecular Biology, UniVersity of Texas Medical School, Houston, Texas 77030, The Photonics Center, Boston UniVersity, Boston, Massachusetts 02215, and Department of Physiology and Biophysics, Boston UniVersity Medical School, Boston, Massachusetts 02118 ReceiVed: May 7, 2007; In Final Form: July 30, 2007 Recent studies demonstrate that photoactive proteins can react within several picoseconds to photon absorption by their chromophores. Faster subpicosecond protein responses have been suggested to occur in rhodopsin- like proteins where retinal photoisomerization may impulsively drive structural changes in nearby protein groups. Here, we test this possibility by investigating the earliest protein structural changes occurring in proteorhodopsin (PR) using ultrafast transient infrared (TIR) spectroscopy with ∼200 fs time resolution combined with nonperturbing isotope labeling. PR is a recently discovered microbial rhodopsin similar to bacteriorhodopsin (BR) found in marine proteobacteria and functions as a proton pump. Vibrational bands in the retinal fingerprint (1175-1215 cm -1 ) and ethylenic stretching (1500-1570 cm -1 ) regions characteristic of all-trans to 13-cis chromophore isomerization and formation of a red-shifted photointermediate appear with a 500-700 fs time constant after photoexcitation. Bands characteristic of partial return to the ground state evolve with a 2.0-3.5 ps time constant. In addition, a negative band appears at 1548 cm -1 with a time constant of 500-700 fs, which on the basis of total- 15 N and retinal C15D (retinal with a deuterium on carbon 15) isotope labeling is assigned to an amide II peptide backbone mode that shifts to near 1538 cm -1 concomitantly with chromophore isomerization. Our results demonstrate that one or more peptide backbone groups in PR respond with a time constant of 500-700 fs, almost coincident with the light-driven retinylidene chromophore isomerization. The protein changes we observe on a subpicosecond time scale may be involved in storage of the absorbed photon energy subsequently utilized for proton transport. Introduction Proteorhodopsin (PR), a newly discovered microbial rhodop- sin found in marine proteobacteria, functions as a light-driven proton pump 1,2 similar to bacteriorhodopsin (BR). Over 4000 different PR variants have been discovered that are distributed throughout the world’s oceans. 3-5 PR-containing bacteria ac- count for ∼13% of the microorganisms in the oceans’ photic zone and are responsible for a significant fraction of the biosphere’s solar energy conversion. 2,6 Similar to BR and other microbial rhodopsins, PR consists of seven transmembrane R-helices containing a retinylidene chromophore covalently bound to a lysine (Lys-231) via a protonated Schiff base. 1 Light absorption by PR results in an all-trans to 13-cis isomerization of the retinal which then drives a series of reaction steps taking a total of approximately 20 ms that result in a proton being pumped from the cytoplasm to the extracellular medium. 7 Recent ultrafast visible absorption experiments 8,9 indicate that the primary photoreaction in PR occurs on a similar time scale (∼0.5 ps) to BR. 10-12 However, visible absorption studies only probe the electronic state of the chromophore. In contrast, IR difference spectroscopy can directly detect small changes occurring in all the molecular components of proteins. 13 Low- temperature (80 K) Fourier transform infrared (FTIR) difference spectroscopy detects changes occurring in the conformation of the chromophore, protein, and internal water molecules of a green-absorbing PR (GPR) during the GPR f K phototransi- tion. 14 However, measurements on cryogenically trapped inter- mediate states may not accurately reflect native structural changes occurring in PR and other proteins on ultrafast time scales at room temperature (RT ) 298 K). Recent ultrafast transient infrared (TIR) studies on photoactive proteins such as photoactive yellow protein, 15 myoglobin, 16 and green fluorescent protein 17 demonstrate that proteins can react within several picoseconds to nanoseconds following photon absorption by their chromophores. UV resonance Raman spectroscopy on rhodopsin 18 and ultrafast TIR measurements on the related archaeal microbial rhodopsins BR, 10 sensory rhodopsin II (SRII), 19 and halorhodopsin (HR) 20 all suggest that protein responses may occur even faster, and may be coincident with the initial subpicosecond all-trans- to 13-cis-retinal isomer- * Corresponding author. E-mail: kjr@bu.edu. † Department of Physics, Boston University. ‡ Department of Chemistry, Boston University. § Department of Biomedical Engineering, Boston University. | University of Texas Medical School. ⊥ The Photonics Center, Boston University. ∇ Boston University Medical School. 11824 J. Phys. Chem. B 2007, 111, 11824-11831 10.1021/jp073490r CCC: $37.00 © 2007 American Chemical Society Published on Web 09/19/2007