title: Spectroscopic and kinetic characterization of the light-dependent enzyme protochlorophyllide oxidoreductase (POR) using monovinyl and divinyl substrates.
abstract: The enzyme POR [Pchlide (protochlorophyllide) oxidoreductase] catalyses the reduction of Pchlide to chlorophyllide, which is a key step in the chlorophyll biosynthesis pathway. This light-dependent reaction has previously been studied in great detail but recent reports suggest that a mixture of MV (monovinyl) and DV (divinyl) Pchlides may have influenced some of these properties of the reaction. Low-temperature absorbance and fluorescence spectroscopy have revealed several spectral differences between MV and DV Pchlides, which were purified from a Rhodobacter capsulatus strain that was shown to contain a mixture of the two pigments. A thorough steady-state kinetic characterization using both Pchlide forms demonstrates that neither pigment appears to affect the kinetic properties of the enzyme. The reaction has also been monitored following illumination at low temperatures and was shown to consist of an initial photochemical step followed by four 'dark' steps for both pigments. However, minor differences were observed in the spectral properties of some of the intermediates, although the temperature dependency of each step was nearly identical for the two pigments. This work provides the first detailed kinetic and spectroscopic study of this unique enzyme using biologically important MV and DV substrate analogues. It also has significant implications for the DV reductase enzyme, which is responsible for converting DV pigments into their MV counterparts, and its position in the sequence of reactions that comprise the chlorophyll biosynthesis pathway.
authors.affiliation: Robert Hill Institute for Photosynthesis and Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK. derren.heyes@manchester.ac.uk
authors.forename: Derren J
authors.initials: DJ
authors.lastname: Heyes
authors.forename: Jerzy
authors.initials: J
authors.lastname: Kruk
authors.forename: C Neil
authors.initials: CN
authors.lastname: Hunter
mesh_terms: D002734:Chlorophyll / Q000096:biosynthesis; D007700:Kinetics; D008027:Light; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry* / Q000378:metabolism* / Q000528:radiation effects; D011521:Protochlorophyllide / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D016354:Rhodobacter capsulatus / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D002734:Chlorophyll; D011521:Protochlorophyllide; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase