Multiple active site residues are important for photochemical efficiency in the light-activated enzyme protochlorophyllide oxidoreductase (POR)

Abstract

Protochlorophyllide oxidoreductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide), an essential, regulatory step in chlorophyll biosynthesis. The unique requirement of the enzyme for light has provided the opportunity to investigate how light energy can be harnessed to power biological catalysis and enzyme dynamics. Excited state interactions between the Pchlide molecule and the protein are known to drive the subsequent reaction chemistry. However, the structural features of POR and active site residues that are important for photochemistry and catalysis are currently unknown, because there is no crystal structure for POR. Here, we have used static and time-resolved spectroscopic measurements of a number of active site variants to study the role of a number of residues, which are located in the proposed NADPH/Pchlide binding site based on previous homology models, in the reaction mechanism of POR. Our findings, which are interpreted in the context of a new improved structural model, have identified several residues that are predicted to interact with the coenzyme or substrate. Several of the POR variants have a profound effect on the photochemistry, suggesting that multiple residues are important in stabilizing the excited state required for catalysis. Our work offers insight into how the POR active site geometry is finely tuned by multiple active site residues to support enzyme-mediated photochemistry and reduction of Pchlide, both of which are crucial to the existence of life on Earth.

Effects of different growth temperatures on growth, development, and plastid pigments metabolism of tobacco (Nicotiana tabacum L.) plants

“…The higher expression of FeCH in leaves also was observed in the study, and this could be ascribed to the reduction of Chls in tobacco leaves under 28.5 °C. At the later steps of Chls biosynthesis in plants, POR catalyzes the conversion of Pchlide into Chlide, accompanying with the degradation of POR and the synthesis of Chls (Beale 1999 , 2005 ; Kim and Apel 2012 ; Talaat 2013 ; Menon et al 2016 ). In the present work, it was observed that 18.5 °C significantly reduced the expression of POR gene in tobacco leaves during 15–30 days (Fig.…”

“…At the later steps of Chls biosynthesis, the light-dependent protochlorophyllide oxidoreductase (POR) catalyzes the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide). In the processes, the tripolymer of the POR-NADPH-Pchlide was disaggregated, and the Chlide was released accompany with the breakdown of POR (Apel et al 1980 ; Armstrong et al 1995 ; Holtorf and Apel 1996 ; Reinbothe et al 2010 ; Kim and Apel 2012 ; Talaat 2013 ; Menon et al 2016 ). Finally, Chlide was catalyzed by chlorophyll synthetase (CHL) and chlorophyllide a oxygenase (CAO), respectively, and then turned into Chl a and Chl b, respectively (Beale 1999 , 2005 ).…”

Dual role of the active site ‘lid’ regions of protochlorophyllide oxidoreductase in photocatalysis and plant development

“…1A) [18]. Although several residues important to photocatalysis are known [23][24][25], a structural basis for POR photocatalysis has emerged only recently. Crystal structures of cyanobacterial POR as the apo-enzyme and a NADPH-bound complex are known [26,27].…”

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

“…[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.…”

“…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.…”