Enzymology below 200 K: The kinetics and thermodynamics of the photochemistry catalyzed by protochlorophyllide oxidoreductase

Abstract

The chlorophyll biosynthesis enzyme protochlorophyllide reductase (POR) catalyzes the light-dependent reduction of protochlorophyllide (Pchlide) into chlorophyllide in the presence of NADPH. As POR is light-dependent, catalysis can be initiated by illumination of the enzyme-substrate complex at low temperatures, making it an attractive model for studying aspects of biological proton and hydride transfers. The early stages in the photoreduction, involving Pchlide binding and an initial photochemical reaction, have been studied in vitro by using low-temperature fluorescence and absorbance measurements. Formation of the ternary POR-NADPHPchlide complex produces red shifts in the fluorescence and absorbance maxima of Pchlide, allowing the dissociation constant for Pchlide binding to be measured. We demonstrate that the product of an initial photochemical reaction, which can occur below 200 K, is a nonfluorescent intermediate with a broad absorbance band at 696 nm (A696) that is suggested to represent an ion radical complex. The temperature dependence of the rate of A696 formation has allowed the activation energy for the photochemical step to be calculated and has shown that POR catalysis can proceed at much lower temperatures than previously thought. Calculations of differences in free energy between various reaction intermediates have been calculated; these, together with the quantum efficiency for Pchlide conversion, suggest a quantitative model for the thermodynamics of the light-driven step of Pchlide reduction.

The First Catalytic Step of the Light-driven Enzyme Protochlorophyllide Oxidoreductase Proceeds via a Charge Transfer Complex

“…The broad absorbance band of the nonfluorescent catalytic intermediate in Fig. 3A is similar to those previously reported (11) and is highly asymmetric, extending as far as 800 nm. The second derivative of this spectrum (Fig.…”

“…The ability to freeze in various catalytic intermediates at low temperatures, in this case the first identifiable intermediate, A 696 , is a crucial step in our understanding of the enzyme mechanism (10 -13). Previous work has provided contradicting reports on the nature of the catalytic intermediate formed after the initial photochemistry, which can occur below 200 K. This nonfluorescent intermediate has a broad absorbance band at 696 nm (11) and has been suggested to represent either a radical species (10,15) or a charge transfer complex (17). We have now used a combination of EPR, ENDOR, Stark, and low temperature absorbance spectroscopy to show that this intermediate is a charge transfer complex formed by hydride transfer from NADPH.…”

“…Although it has been shown that the POR-NADP ϩ -Chlide product complex is only produced after the first dark step (13), the exact molecular nature of the intermediate formed after the initial photochemistry, which is essential to our understanding of the overall catalytic mechanism, remains unclear. The product of this lightdriven step is a nonfluorescent intermediate with a broad absorbance band at 696 nm (A 696 ) (11,15). EPR measurements using etioplast membranes and heterologously expressed protein have suggested that this intermediate is a radical species (10,15).…”

“…PCC6803 was overproduced and purified as previously described (12). Pchlide was isolated from Rhodobacter capsulatus ZY5 cultures as previously described (11 Preparation of Cation Radicals in Vitro-Pchlide and Chlide cation radicals were formed by oxidation of solutions in dry 10:1 dichloromethane:tetrahydrofuran, using a 5-fold excess of resublimed iodine, on a high vacuum line. The samples were transferred anaerobically to quartz EPR tubes and frozen immediately in liquid nitrogen.…”

Temperature-dependent macromolecular X-ray crystallography

“…In addition to its importance as a prominent feature in the low-temperature physics of biological macromolecules, the dynamical transition has been linked to the onset of biological activity (Rasmussen et al, 1992;Lichtenegger et al, 1999;Ostermann et al, 2000). However, certain enzymes are active below the dynamical transition (Daniel et al, 1998), or are at least able to undergo part of their catalytic cycle (Heyes et al, 2002;Durin et al, 2009).…”