title: Enzyme activation and catalysis: characterisation of the vibrational modes of substrate and product in protochlorophyllide oxidoreductase.
abstract: The light-dependent reduction of protochlorophyllide, a key step in the synthesis of chlorophyll, is catalyzed by the enzyme protochlorophyllide oxidoreductase (POR) and requires two photons (O. A. Sytina et al., Nature, 2008, 456, 1001-1008). The first photon activates the enzyme-substrate complex, a subsequent second photon initiates the photochemistry by triggering the formation of a catalytic intermediate. These two events are characterized by different spectral changes in the infra-red spectral region. Here, we investigate the vibrational frequencies of the POR-bound and unbound substrate, and product, and thus provide a detailed assignment of the spectral changes in the 1800-1250 cm(-1) region associated with the catalytic conversion of PChlide:NADPH:TyrOH into Chlide:NADP(+):TyrO(-). Fluorescence line narrowed spectra of the POR-bound Pchlide reveal a C=O keto group downshifted by more than 20 cm(-1) to a relatively low vibrational frequency of 1653 cm(-1), as compared to the unbound Pchlide, indicating that binding of the chromophore to the protein occurs via strong hydrogen bond(s). The frequencies of the C=C vibrational modes are consistent with a six-coordinated state of the POR-bound Pchlide, suggesting that there are two coordination interactions between the central Mg atom of the chromophore and protein residues, and/or a water molecule. The frequencies of the C=C vibrational modes of Chlide are consistent with a five-coordinated state, indicating a single interaction between the central Mg atom of the chromophore and a water molecule. Rapid-scan FTIR measurements on the Pchlide:POR:NADPH complex at 4 cm(-1) spectral resolution reveal a new band in the 1670 cm(-1) region. The FTIR spectra of the enzyme activation phase indicate involvement of a nucleotide-binding structural motif, and an increased exposure of the protein to solvent after activation.
authors.affiliation: Department of Physics and Astronomy, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands. sytina@amolf.nl
authors.forename: Olga A
authors.initials: OA
authors.lastname: Sytina
authors.forename: Maxime T
authors.initials: MT
authors.lastname: Alexandre
authors.forename: Derren J
authors.initials: DJ
authors.lastname: Heyes
authors.forename: C Neil
authors.initials: CN
authors.lastname: Hunter
authors.forename: Bruno
authors.initials: B
authors.lastname: Robert
authors.forename: Rienk
authors.initials: R
authors.lastname: van Grondelle
authors.forename: Marie Louise
authors.initials: ML
authors.lastname: Groot
mesh_terms: D002384:Catalysis; D004789:Enzyme Activation; D007700:Kinetics; D008027:Light; D009249:NADP / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry* / Q000378:metabolism*; D010777:Photochemistry; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D009249:NADP; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase