Laser Excitation Studies of the Product Release Steps in the Catalytic Cycle of the Light-driven Enzyme, Protochlorophyllide Oxidoreductase

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 ϳ72 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.

Nuclear Quantum Tunneling in the Light-activated Enzyme Protochlorophyllide Oxidoreductase

“…Laser Photoexcitation Measurements-Photoexcitation of POR ternary complexes (POR-NADPH-Pchlide) was by excitation at 450 nm, using an optical parametric oscillator of a Q-switched Nd-YAG laser (Brilliant B, Quantel) in a cuvette of 1-cm path length as described (15). Laser pulses (ϳ60 mJ) were between 6 and 8 ns in duration.…”

“…1), a finding based on previous site-directed mutagenesis studies (17) and the structural homology model of the Synechocystis enzyme (18). The remaining dark reactions involve a series of ordered product release and cofactor binding steps, which are linked to conformational changes in the enzyme (13)(14)(15).…”

“…However, despite its biological significance, the detailed mechanism of this light-activated catalysis remains poorly understood. Low temperature spectroscopy has identified a number of steps in the reaction cycle, including an initial light-driven reaction (11,12) and subsequent dark reactions (13)(14)(15). In the reaction, reduction of Pchlide by NADPH in the POR-NADPH-Pchlide complex involves hydride transfer from the pro-S face of NADPH to the C-17 atom of Pchlide (16).…”

A Twin-track Approach Has Optimized Proton and Hydride Transfer by Dynamically Coupled Tunneling during the Evolution of Protochlorophyllide Oxidoreductase

“…The formation of an intermediate with an absorbance band at 696 nm represents an initial hydride transfer reaction from NADPH, and the subsequent slower exponential decay in absorbance at 696 nm represents the proton transfer reaction (12). Slower kinetically resolved absorbance changes at 670 nm on the millisecond time scale represent a series of ordered product release and cofactor rebinding events (18). For studies of the temperature dependence of rate constants, data were fitted to the Eyring equation.…”

“…Proton transfer is therefore reliant on an extended network of molecular motions coupled to solvent dynamics, whereas hydride transfer is linked only to localized motions within the enzyme active site. Following hydride and proton transfer, the catalytic cycle is completed by a series of ordered product release and cofactor binding steps, which are also linked to major conformational changes in the enzyme (18).…”