Synthesis of 4R- and 4S-tritium labeled NADPH for the determination of the coenzyme stereospecificity of NADPH: Protochlorophyllide oxidoreductase

Valera, Vimlesh; Fung, Margaret; Wessler, A.; Richards, William R.

doi:10.1016/0006-291x(87)91141-7

Cited by 42 publications

(29 citation statements)

References 8 publications

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“…The hydride is transferred from the pro-S face of the nicotinamide ring to the C-17 position of the Pchlide molecule (6,7), and a conserved Tyr residue has been proposed to donate a proton to the C-18 position. The close proximity of a conserved Lys residue is thought to be necessary to lower the apparent pK a of the phenolic group of the Tyr, allowing deprotonation to occur (2).…”

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confidence: 99%

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

Heyes

Heathcote

Rigby

et al. 2006

Journal of Biological Chemistry

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In chlorophyll biosynthesis protochlorophyllide reductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide) to chlorophyllide, providing a rare opportunity to trap and characterize catalytic intermediates at low temperatures. Moreover, the presence of a chlorophyll-like molecule allows the use of EPR, electron nuclear double resonance, and Stark spectroscopies, previously used for the analysis of photosynthetic systems, to follow catalytic events in the active site of POR. Different models involving the formation of either radical species or charge transfer complexes have been proposed for the initial photochemical step, which forms a nonfluorescent intermediate absorbing at 696 nm (A 696 ). Our EPR data show that the concentration of the radical species formed in the initial photochemical step is not stoichiometric with conversion of substrate. Instead, a large Stark effect, indicative of charge transfer character, is associated with A 696 . Two components were required to fit the Stark data, providing clear evidence that charge transfer complexes are formed during the initial photochemistry. The temperature dependences of both A 696 formation and NADPH oxidation are identical, and we propose that formation of the A 696 state involves hydride transfer from NADPH to form a charge transfer complex. A catalytic mechanism of POR is suggested in which Pchlide absorbs a photon, creating a transient charge separation across the C-17-C-18 double bond, which promotes ultrafast hydride transfer from the pro-S face of NADPH to the C-17 of Pchlide. The resulting A 696 charge transfer intermediate facilitates transfer of a proton to the C-18 of Pchlide during the subsequent first "dark" reaction.

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confidence: 99%

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

Heyes

Heathcote

Rigby

et al. 2006

Journal of Biological Chemistry

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“…Following light activation of this complex, a hydride ion is transferred from the pro-S face of NADPH to the C-17 atom of Pchlide (6,7). The valence of the C-18 atom is satisfied by proton transfer, which is suggested to originate from an active site tyrosine residue (8).…”

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Cryogenic and Laser Photoexcitation Studies Identify Multiple Roles for Active Site Residues in the Light-driven Enzyme Protochlorophyllide Oxidoreductase

Menon

Waltho

Scrutton

et al. 2009

Journal of Biological Chemistry

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The light-activated enzyme NADPH-protochlorophyllide oxidoreductase (POR) catalyzes the trans addition of hydrogen across the C-17-C-18 double bond of protochlorophyllide (Pchlide), a key step in chlorophyll biosynthesis. Similar to other members of the short chain alcohol dehydrogenase/reductase family of enzymes, POR contains a conserved Tyr and Lys residue in the enzyme active site, which are implicated in a proposed reaction mechanism involving proton transfer from the Tyr hydoxyl group to Pchlide. We have analyzed a number of POR variant enzymes altered in these conserved residues using a combination of steady-state turnover, laser photoexcitation studies, and low temperature fluorescence spectroscopy. None of the mutations completely abolished catalytic activity. We demonstrate their importance to catalysis by defining multiple roles in the overall reaction pathway. Mutation of either residue impairs formation of the ground state ternary enzyme-substrate complex, pointing to a key role in substrate binding. By analyzing the most active variant (Y193F), we show that Tyr-193 participates in proton transfer to Pchlide and stabilizes the Pchlide excited state, enabling hydride transfer from NADPH to Pchilde. Thus, in addition to confirming the probable identity of the proton donor in Pchlide reduction, our work defines additional roles for these residues in facilitating hydride transfer through stabilization of the ground and excited states of the ternary enzyme complex.

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“…The reaction catalyzed by POR involves hydride transfer from the pro-S face of NADPH to the C-17 position of the Pchlide molecule (10,11). It has been proposed that the conserved Tyr donates a proton to the C18 position with the Lys residue thought to be important for lowering the apparent pK a of the Tyr, allowing deprotonation to occur (5).…”

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confidence: 99%

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

Heyes

Ruban

Wilks

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

141

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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.

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Synthesis of 4R- and 4S-tritium labeled NADPH for the determination of the coenzyme stereospecificity of NADPH: Protochlorophyllide oxidoreductase

Cited by 42 publications

References 8 publications

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

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

Cryogenic and Laser Photoexcitation Studies Identify Multiple Roles for Active Site Residues in the Light-driven Enzyme Protochlorophyllide Oxidoreductase

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

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