Photoisomerization and Proton Transfer in Photoactive Yellow Protein Michael J. Thompson, Donald Bashford,* Louis Noodleman,* and Elizabeth D. Getzoff* Department of Molecular Biology, MB4, Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, California 92037 Received November 22, 2002; E-mail: bashford@scripps.edu; lou@scripps.edu; edg@scripps.edu Abstract: The photoactive yellow protein (PYP) is a bacterial photosensor containing a para-coumaryl thioester chromophore that absorbs blue light, initiating a photocycle involving a series of conformational changes. Here, we present computational studies to resolve uncertainties and controversies concerning the correspondence between atomic structures and spectroscopic measurements on early photocycle intermediates. The initial nanoseconds of the PYP photocycle are examined using time-dependent density functional theory (TDDFT) to calculate the energy profiles for chromophore photoisomerization and proton transfer, and to calculate excitation energies to identify photocycle intermediates. The calculated potential energy surface for photoisomerization matches key, experimentally determined, spectral parameters. The calculated excitation energy of the photocycle intermediate cryogenically trapped in a crystal structure by Genick et al. [Genick, U. K.; Soltis, S. M.; Kuhn, P.; Canestrelli, I. L.; Getzoff, E. D. Nature 1998, 392, 206-209] supports its assignment to the PYPB (I0) intermediate. Differences between the time-resolved room temperature (298 K) spectrum of the PYPB intermediate and its low temperature (77 K) absorbance are attributed to a predominantly deprotonated chromophore in the former and protonated chromophore in the latter. This contrasts with the widely held belief that chromophore protonation does not occur until after the PYP L (I1 or pR) intermediate. The structure of the chromophore in the PYPL intermediate is determined computationally and shown to be deprotonated, in agreement with experiment. Calculations based on our PYP B and PYPL models lead to insights concerning the PYPBL intermediate, observed only at low temperature. The results suggest that the proton is more mobile between Glu46 and the chromophore than previously realized. The findings presented here provide an example of the insights that theoretical studies can contribute to a unified analysis of experimental structures and spectra. 1 Introduction Many photosensory proteins transduce the energy of an absorbed photon into a signal to initiate a cellular response via a photocycle, which is a sequence of coupled electronic and conformational changes in a protein-chromophore system. Newly developed time-resolved and cryogenic trapping tech- niques in both spectroscopy and structural biology have gener- ated a wealth of data about the function of photoactive proteins and the nature of their photocycle intermediates. However, coupling the results from these diverse techniques to develop a coherent picture of protein function remains a challenge. Here, we use quantum chemical calculations to resolve questions from experimental photobiology. The photoactive yellow protein (PYP) is among the most well-characterized photosensors. Its small size (14 kD), high thermal stability and water solubility, and the similarity of its photocycle with the bacterial rhodopsins, 1-3 make it an attractive model system for biological photosensing. PYP exists in several halophilic purple bacteria, 4-6 where it is thought to serve as the photosensor initiating negative phototactic response to blue light. 7 PYP’s chromophore, p-coumaric acid, is attached via a thioester linkage to the only cysteine residue in the protein (Cys69). 2,8,9 In the dark state of the photocycle, the chromophore is in the trans conformation 9 and is deprotonated. 8-11 The buried charge on the chromophore is stabilized by hydrogen bonds from Tyr42 and Glu46 (protonated) to its phenolate oxygen (Figure 1a), and from the peptide backbone to its carbonyl oxygen (not shown). 9 Following the absorption of blue light (λ max ) 446 (1) Meyer, T. E.; Yakali, E.; Cusanovich, M. A.; Tollin, G. Biochemistry 1987, 26, 418. (2) Hoff, W. D.; Dux, P.; Hard, K.; Devreese, B.; Nugteren-Roodzant, I. M.; Crielaard, W.; Boelens, R.; Kaptein, R.; van Beeumen., J.; Hellingwerf, K. J. Biochemistry 1994, 33, 13 959. (3) Hellingwerf, K. J.; Hoff, W. D.; Crielaard, W. Mol. 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