Abstract
The latter stages of the catalytic cycle of the light-driven enzyme, protochlorophyllide oxidoreductase, have been investigated using novel laser photoexcitation methods. The formation of the ternary product complex was initiated with a 6-ns laser pulse, which allowed the product release steps to be kinetically accessed for the first time. Subsequent absorbance changes associated with the release of the NADP+ and chlorophyllide products from the enzyme could be followed on a millisecond timescale. This has facilitated a detailed kinetic and thermodynamic characterization for the interconversion of all the various bound and unbound product species. Initially, NADP+ is released from the enzyme in a biphasic process with rate constants of 1210 and 237 s(-1). The rates of both phases show a significant dependence on the viscosity of the solvent and become considerably slower at higher glycerol concentrations. The fast phase of this process exhibits no dependence on NADP+ concentration, suggesting that conformational changes are required prior to NADP+ release. Following NADP+ release, the NADPH rebinds to the enzyme with a maximum rate constant of approximately 72 s(-1). At elevated temperatures (>298 K) chlorophyllide is released from the enzyme to yield the free product with a maximum rate constant of 20 s(-1). The temperature dependencies of the rates of each of these steps were measured, and enthalpies and entropies of activation were calculated using the Eyring equation. A comprehensive kinetic and thermodynamic scheme for these final stages of the reaction mechanism is presented.
Highlights
NOVEMBER 2, 2007 VOLUME 282 NUMBER 44 alyzes the light-dependent trans addition of hydrogen across the C17ϭC18 double bond of the D-ring of protochlorophyllide (Pchlide) to produce chlorophyllide (Chlide), which is a key reaction within the chlorophyll biosynthesis pathway and the subsequent assembly of the photosynthetic apparatus (Fig. 1A) [1,2,3,4]
Catalysis has been triggered using a 6-ns laser pulse, leading to the formation of the ternary protochlorophyllide oxidoreductase (POR)-NADPϩ-Chlide product complex and facilitating the measurement of absorbance changes associated with the release of NADPϩ and Chlide from the enzyme
Kinetic Overview of the Product Release Steps—The catalytic reaction of POR has been triggered by using a 6-ns laser pulse tuned to the Soret region of the Pchlide absorbance spectrum
Summary
Sample Preparation—Recombinant POR from the thermophilic cyanobacterium Thermosynechococcus elongatus was overexpressed in Escherichia coli and purified as described previously [8]. Laser Flash Photolysis—For laser photoexcitation experiments, 1-ml samples containing 30 M POR, 100 M NADPH, and 10 M Pchlide in 50 mM Tris-HCl, pH 7.5, 0.1% Genapol, 0.1% -mercaptoethanol were excited at 450 nm by using an optical parametric oscillator of a Q-switched Nd-YAG laser (Brilliant B, Quantel) in a cuvette of 1-cm path length. Absorbance spectra were measured before and after laser photoexcitation by transferring the cuvette to a Cary 50 UV/visible spectrophotometer (Varian) and recording data between 300 – 800 nm. In temperature dependence studies the kinetic and thermodynamic parameters were obtained by fitting the data to the Eyring equation as described previously [14, 15]
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