Identification and characterization of the product release steps within the catalytic cycle of protochlorophyllide oxidoreductase.
The chlorophyll biosynthetic enzyme protochlorophyllide reductase (POR) catalyzes the reduction of protochlorophyllide (Pchlide) into chlorophyllide (Chlide) with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. POR is a light-driven enzyme, which has provided a unique opportunity to trap intermediates and identify different steps in the reaction pathway by initiating catalysis with illumination at low temperatures. In the present work we have used a thermophilic form of POR, which has an increased conformational rigidity at comparable temperatures, to dissect and study the final stages of the reaction where protein dynamics are proposed to play an important role in catalysis. Low-temperature fluorescence and absorbance measurements have been used to demonstrate that the reaction pathway for this enzyme consists of two additional "dark" steps, which have not been detected in previous studies. Product binding studies were used to show that spectroscopically distinct Chlide species could be observed and were dependent on whether the NADPH or NADP(+) cofactor was present. As a result we have been able to identify the intermediates that are observed during the latter stages of the POR catalytic cycle and have shown that they are formed via a series of ordered product release and cofactor binding events. These events involve release of NADP(+) from the enzyme and its replacement by NADPH, before release of the Chlide product has taken place. Following release of Chlide, the subsequent binding of Pchlide allows the next catalytic cycle to proceed.
Heyes
Derren J
DJ
Robert Hill Institute for Photosynthesis, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, United Kingdom. d.j.heyes@shef.ac.uk
Hunter
C Neil
CN
D002384:Catalysis; D002735:Chlorophyllides / Q000737:chemistry / Q000378:metabolism*; D000458:Cyanobacteria / Q000201:enzymology / Q000235:genetics; D003624:Darkness; D004926:Escherichia coli / Q000378:metabolism; D008027:Light; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics / Q000378:metabolism*; D010777:Photochemistry; D011521:Protochlorophyllide / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature
D002735:Chlorophyllides; D011994:Recombinant Proteins; D011521:Protochlorophyllide; D009249:NADP; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase