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Issue 2, 2012
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Spectroscopic characterization of the first ultrafast catalytic intermediate in protochlorophyllide oxidoreductase

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Abstract

The enzyme NADPH:protochlorophyllide oxidoreductase (POR) catalyses the reduction of protochlorophyllide into chlorophyllide, a precursor of chlorophyll a in photosynthetic organisms. The enzyme binds the substrate and the cofactor in the dark and catalysis is initiated by the absorption of light by the substrate. We have carried out spectroscopic measurements with ultrafast time resolution under single pulse conditions, which reveal a biphasic formation of the first catalytic intermediate, I675* with average rates of (3.7 ± 0.7 ps)−1 and (177 ± 78 ps)−1, as obtained from a systematic analysis of 15 datasets. Measurements in the mid-IR absorption spectral region show that I675* is associated with a decrease of the PChlide C[double bond, length as m-dash]O keto oscillator strength. The spectroscopic changes in the visible and mid-IR regions are specific for the enzyme reaction as they do not occur in the photoexcited substrate alone. In deuterated samples, the rates of I675* formation are reduced by a factor of 1.5–2 compared to protonated samples, suggesting the involvement of a proton movement in this reaction step. The quantum yield of this step is determined to be 0.64 ± 0.11, and the quantum yield of the final reaction product formed on a later time scale, chlorophyllide, is 0.26 ± 0.06. Several possible interpretations of these data are discussed.

Graphical abstract: Spectroscopic characterization of the first ultrafast catalytic intermediate in protochlorophyllide oxidoreductase

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Publication details

The article was received on 26 May 2011, accepted on 25 Oct 2011 and first published on 28 Nov 2011


Article type: Paper
DOI: 10.1039/C1CP21713E
Citation: Phys. Chem. Chem. Phys., 2012,14, 616-625
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    Spectroscopic characterization of the first ultrafast catalytic intermediate in protochlorophyllide oxidoreductase

    O. A. Sytina, I. H. M. van Stokkum, D. J. Heyes, C. N. Hunter and M. L. Groot, Phys. Chem. Chem. Phys., 2012, 14, 616
    DOI: 10.1039/C1CP21713E

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