Identification and Characterization of the Product Release Steps within the Catalytic Cycle of Protochlorophyllide Oxidoreductase

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• 作者：Derren J. Heyes and C. Neil Hunter
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：June 29, 2004
• 年：2004
• 卷：43
• 期：25
• 页码：8265 - 8271
• 全文大小：155K
• 年卷期：v.43,no.25(June 29, 2004)
• ISSN：1520-4995

文摘

The chlorophyll biosynthetic enzyme protochlorophyllide reductase (POR) catalyzes thereduction of protochlorophyllide (Pchlide) into chlorophyllide (Chlide) with reduced nicotinamide adeninedinucleotide phosphate (NADPH) as a cofactor. POR is a light-driven enzyme, which has provided aunique opportunity to trap intermediates and identify different steps in the reaction pathway by initiatingcatalysis with illumination at low temperatures. In the present work we have used a thermophilic form ofPOR, which has an increased conformational rigidity at comparable temperatures, to dissect and studythe 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 thereaction pathway for this enzyme consists of two additional "dark" steps, which have not been detectedin previous studies. Product binding studies were used to show that spectroscopically distinct Chlidespecies 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 ofthe POR catalytic cycle and have shown that they are formed via a series of ordered product release andcofactor binding events. These events involve release of NADP⁺ from the enzyme and its replacement byNADPH, before release of the Chlide product has taken place. Following release of Chlide, the subsequentbinding of Pchlide allows the next catalytic cycle to proceed.