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

Abstract

Hydride transfer plays a crucial role in a wide range of biological systems. However, its mode of action (concerted or stepwise) is still under debate. Light‐dependent NADPH: protochlorophyllide oxidoreductase (POR) catalyzes the stereospecific trans addition of a hydride anion and a proton across the C17−C18 double bond of protochlorophyllide. Time‐resolved absorption and emission spectroscopy were used to investigate the hydride transfer mechanism in POR. Apart from excited states of protochlorophyllide, three discrete intermediates were resolved, consistent with a stepwise mechanism that involves an initial electron transfer from NADPH. A subsequent proton‐coupled electron transfer followed by a proton transfer yield distinct different intermediates for wild type and the C226S variant, that is, initial hydride attaches to either C17 or C18, but ends in the same chlorophyllide stereoisomer. This work provides the first evidence of a stepwise hydride transfer in a biological system.

Preise der Royal Society of Chemistry 2019

“…Scruttons Gruppe untersucht Grundlagen und Anwendungen der biologischen Katalyse sowie die Umsetzung biophysikalisch‐mechanistischer Studien in Synthesebiologie und Biotechnologie. In einer Angewandte‐Chemie ‐Zuschrift gaben sie Einblicke in den Reaktionsmechanismus der lichtabhängigen Protochlorophyllid‐Oxidoreduktase …”

Extending the Library of Light‐Dependent Protochlorophyllide Oxidoreductases and their Solvent Tolerance, Stability in Light and Cofactor Flexibility

“…LPORs are found in different phototrophic organisms [14] and have been described as single component NADPH-dependent enzymes catalyzing the light-driven reduction of a C=C double bond at ring D of protochlorophyllide (pchlide) to chlorophyllide (chlide), which is a key intermediate in the biosynthesis of chlorophyll (Scheme 1). [15] The reduction of the C17=C18 double bond occurs in a sequential manner by trans-addition of a hydride and a proton along the double bond, [14,16] after excitation of the pchlide by light. [16][17] Actually, first a ternary complex of substrate, cofactor and enzyme forms, which acts as a photoreceptor during the reaction.…”

“…[15] The reduction of the C17=C18 double bond occurs in a sequential manner by trans-addition of a hydride and a proton along the double bond, [14,16] after excitation of the pchlide by light. [16][17] Actually, first a ternary complex of substrate, cofactor and enzyme forms, which acts as a photoreceptor during the reaction. Under illumination, an endergonic light-driven hydride transfer takes place from NADPH to the C17 position of the pchlide, whereby a charge-transfer complex is formed.…”

“…Finally, the NADP + is released from the enzyme and after binding of NADPH the product (chlide) is released and is replaced by another pchlide. [16,18] The whole reaction takes places on an ultrafast timescale and the light-driven step proceeds even under low temperatures of 120 K. [18b,19] Recently, a C226S variant of a LPOR has been found showing a different mechanism. The reaction starts with a single electron transfer followed by a concerted hydrogen atom and proton transfer.…”

“…The exact influence of the cysteine residue within the catalytic mechanism is not fully understood, yet. [17,[20][21] In the last decade a few LPORs were mostly studied from the viewpoint of their photochemical mechanism, [16] especially the LPORs from Synechocystis sp. [14,22] and Thermosynechococcus elongatus [20] were investigated regarding their expression, kinetic parameters and reaction intermediates.…”