Mutagenesis Alters the Catalytic Mechanism of the Light-driven Enzyme Protochlorophyllide Oxidoreductase

Abstract

The light-activated enzyme protochlorophyllide oxidoreductase (POR) catalyzes an essential step in the synthesis of the most abundant pigment on Earth, chlorophyll. This unique reaction involves the sequential addition of a hydride and proton across the C17‫؍‬C18 double bond of protochlorophyllide (Pchlide) by dynamically coupled quantum tunneling and is an important model system for studying the mechanism of hydrogen transfer reactions. In the present work, we have combined site-directed mutagenesis studies with a variety of sensitive spectroscopic and kinetic measurements to provide new insights into the mechanistic role of three universally conserved Cys residues in POR. We show that mutation of Cys-226 dramatically alters the catalytic mechanism of the enzyme. In contrast to wild-type POR, the characteristic charge-transfer intermediate, formed upon hydride transfer from NADPH to the C17 position of Pchlide, is absent in C226S variant enzymes. This suggests a concerted hydrogen transfer mechanism where proton transfer only is rate-limiting. Moreover, Pchlide reduction does not require the network of solvent-coupled conformational changes that play a key role in the proton transfer step of wild-type POR. We conclude that this globally important enzyme is finely tuned to facilitate efficient photochemistry, and the removal of a key interaction with Pchlide in the C226S variants significantly affects the local active site structure in POR, resulting in a shorter donoracceptor distance for proton transfer.The light-activated enzyme protochlorophyllide oxidoreductase (POR) 3 catalyzes the reduction of protochlorophyllide (Pchlide) (Fig. 1A), a key reaction in the chlorophyll biosynthetic pathway that triggers a profound transformation in plant development (1-3). Although the reaction can also be catalyzed by a light-independent Pchlide reductase in non-flowering land plants, algae, and cyanobacteria, higher plants only contain POR and are therefore completely reliant on light for the greening process (1, 2). The light dependence of POR presents a unique opportunity to study catalysis at low temperatures and on ultrafast timescales, both of which are usually inaccessible for the majority of enzymes (1). Recent advances in our understanding of the catalytic mechanism of POR illustrate why it is an important generic model for studying enzyme catalysis and reaction dynamics (4 -12).Low temperature spectroscopy indicates that the catalytic cycle of the enzyme comprises an initial light-driven reaction that involves hydride transfer from the pro-S face of NADPH to the C17 of Pchlide to form a charge-transfer complex (4, 5). This facilitates the subsequent protonation of the C18 position of the Pchlide molecule from a conserved Tyr residue during the first of the dark reactions (5-7). By using laser photoexcitation studies, we have shown that these two sequential enzymatic H-transfer reactions occur by quantum mechanical tunneling (8). Proton tunneling is coupled to promoting motions in the enzyme that facilitate the re...

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

“…The NADPH-binding site is highly conserved in all PORs (Fig. 2A), and single residue changes in this region of the enzyme are known to compromise hydride transfer by substantially affecting the lifetime of the photoexcited state (15,33,34). This is consistent with the geometry and active site dynamics being optimally configured in all POR enzymes to enable efficient photochemistry.…”

“…The reaction chemistry is controlled by localized dynamics in all PORs through conser- vation of protein structure in the enzyme active site. The stringent structural requirements of the hydride transfer chemistry are consistent with the known sensitivity of this reaction to minor active site structural perturbations (15,33,34).…”

Stepwise Hydride Transfer in a Biological System: Insights into the Reaction Mechanism of the Light‐Dependent Protochlorophyllide Oxidoreductase

“…Finally, we investigated the ternary complex, PChlide/POR‐C226S/NADPH, which was previously proposed to proceed via an alternative reaction mechanism compared to wild type (WT) 15. Here, 1 P 1 is quenched from 4.4 ns to 760 ps, resulting in a quantum yield of 83 % (see argument for WT data above) for the first eT forming 2 I 0 (1) which is identical to the situation in WT.…”

Response to the Comments by L. O. Björn on our Paper “Catalytic Efficiency of a Photoenzyme—An Adaptation to Natural Light Conditions”

“…[1][2][3][4][5][6][7] In addition, the thermodynamics of substrate/coenzyme binding as well as the complete POR catalytic cycle have been investigated by spectroscopic techniques in conjunction with low-temperature and stopped flow methods. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] The reason is that such minimal photoactive POR complexes consisting of the natural constituents-photoenzyme, substrate, and cofactor-should allow a more precise insight into the molecular mechanism of POR.As outlined in the supplement of our paper, the POR enzymes used in our experiments are monomers and were obtained from heterologous expression of the POR A and POR B gen from barley (Hordeum vulgare) and the POR gen from Synechocystsis. [8][9][10][11][12][13][14][15][16][17][18] As in the studies cited above, [1][2][3][4][5][6][7][8][9]…”

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] The reason is that such minimal photoactive POR complexes consisting of the natural constituents-photoenzyme, substrate, and cofactor-should allow a more precise insight into the molecular mechanism of POR. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] The reason is that such minimal photoactive POR complexes consisting of the natural constituents-photoenzyme, substrate, and cofactor-should allow a more precise insight into the molecular mechanism of POR.…”