Resonance Raman evidence that the thioester-linked 4-hydroxycinnamoyl chromophore of photoactive yellow protein is deprotonated in the dark state. Kim, M.; Mathies, R.A.; Hoff, W.D.; Hellingwerf, K.J. Published in: Biochemistry DOI:10.1021/bi00039a024Link to publication Citation for published version (APA):Kim, M., Mathies, R. A., Hoff, W. D., & Hellingwerf, K. J. (1995). Resonance Raman evidence that the thioesterlinked 4-hydroxycinnamoyl chromophore of photoactive yellow protein is deprotonated in the dark state. Biochemistry, 34, 12669-12672. DOI: 10.1021/bi00039a024 General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. ABSTRACT: Resonance Raman spectra of the ground state of photoactive yellow protein (PYP), a photoactive pigment found in Ectothiorhodospira halophila, have been obtained with excitation at 413.1 nm using a microspinning sample cell. The resonance Raman spectra of the thioester-linked 4-hydroxycinnamyl chromophore in the protein are compared with the preresonance Raman spectra of the 4-hydroxycinnamyl phenyl thioester and 4-hydroxycinnamic acid model compounds at various pH values. Bands at 1568, 1542, 1500, 1434, and 1166 cm-' in the Raman spectrum of the anionic form of the 4-hydroxycinnamyl phenyl thioester are shown to be characteristic for the deprotonation of the chromophore. The observation of bands in PYP exhibiting very similar frequency and intensity patterns provides strong evidence that the chromophore in PYP is stabilized as a phenolate anion at pH 7.4, in support of conclusions from crystallographic studies. Furthermore, the insensitivity of the PYP Raman spectrum to placement of the protein in D20 buffer is consistent with the absence of the exchangeable phenolic proton on the cinnamyl chromophore. These results establish the feasibility of elucidating the molecular mechanism of light-toinformation transduction by this new photosensory pigment with resonance Raman spectroscopy.Photoactive yellow protein (PYP),' a water-soluble yellowcolored protein with a visible absorption maximum at 446 nm, is found in several halophilic phototrophic bacteria including Ectothiorhodospira halophila (Meyer, 1985;Meyer et al., 1990;Hoff et al., 1994~). E. halophila exhibits a negative phototactic reaction or repellent response to physiological intensiti...

A time-resolved step-scan FTIR spectrometer with a time resolution of 20 ns was developed and used to investigate the KL to L transition in the photocycle of bacteriorhodopsin in the time range from -60 to 940 ns. Broadband FTIR absorbance difference spectra with a spectral range of 850-2050 cm -1 and a spectral resolution of 4 cm -1 have been obtained. Our data show that there are two sets of BR photoproduct difference bands exhibiting different kinetics. The intensity changes of bands attributed to structural changes of the carboxyl groups Asp-96 and Asp-115 as well as of bands assigned to CdC and CsC stretching modes of the chromophore show single exponential behavior with a time constant of about 400 ns, implying that these two processes are coupled. The time-dependent intensity changes of bands attributed to structural changes of the chromophore region near the Schiff base exhibit slower kinetics with a time constant of about 2 µs. We interpret our data in terms of a process during the KL to L transition where structural changes of the -ionone ring end of the chromophore and of Asp-115 occur faster than changes at the Schiff base region of the chromophore. Under our physiological sample conditions, a perturbation of Asp-115 occurs in the first 20 ns in contrast to results from hydrated films where this process is blocked or occurs more slowly. This fast protein response indicates that there is direct coupling between the carboxylic acid residues and the chromophore. Comparison of our data with low-temperature and microsecond time-resolved infrared spectra of the L intermediate in hydrated films of BR indicates that a different L structure is produced when the water activity is low.

No abstract

We describe the performance of a high-throughput system for detecting Raman spectra excited in the range from 320 to 750 nm. The system utilizes notch filters to reject Rayleigh scattering, a single polychromator as the dispersive element, and photon detection via a charge-coupled device. The filters, mirrors, and grating are changed to maximize performance in each excitation region. The excitation source consists of a Kr+ ion laser. Good-quality Raman data are reported for rhodamine 6G at 0.1 mM in methanol with the use of 752.5-nm excitation, and 0.2 mM flavin adenine dinucleotide (FAD) in tris buffer with the use of 647.1-nm excitation. High-quality resonance Raman data for 0.1 mM N-acetyl-glycine ethyl dithio ester in 5% CH3CN/H2O are also reported with the use of 324.0-nm excitation.