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

Abstract

NADPH:protochlorophyllide oxidoreductase (POR) is one of only two enzymes in nature, in which the enzymatic activity is switched on by the absorption of light. The pre-formation of the enzyme-substrate complex in the dark and the initiation of catalysis by light make the POR enzyme an excellent model for studying the initial, ultrafast steps of enzymatic reactions in real time. Throughout the last decade a series of such studies have been reported. They all include recombinant POR enzymes, which were expressed in Escherichia coli and reconstituted to the ternary enzyme complexes by addition of the substrate and coenzyme, protochlorophyllide (PChlide) and NADPH, respectively. [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. All these experiments were also performed on recombinant enzymes expressed in Escherichia coli or on a pigment-free, monomeric enzyme isolated from etiolated oat (Avena sativa) seedlings. [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][10][11][12][13][14][15][16][17][18] we have used purified recombinant POR enzymes in our experiments aimed to determine the quantum yield for the POR catalyzed photoreduction. In the article of L. O. Bjçrn the results obtained from those enzymes are subjected to criticisms in so far as they would differ from those published more than 40 years ago. However, most of the differences can be explained by the different types and aggregation states of the enzymes examined. In the work referred by L. O. Bjçrn, POR bound to natural membranes and POR isolated from etiolated plant material was investigated. [19][20][21] There is strong evidence that POR associates with membranes under natural conditions and exists in an aggregated state as dimer or larger sized oligomer. [22] In a similar manner, the enzymes obtained from the extraction of etiolated plant material, PChlide holochromes, form high molecular weight complexes (~600 kDa), too. [19,21,23] The photochemistry of aggregated PChlide-POR complexes is impaired by pigment-pigment interactions, energy transfer between PChlide and chlorophyllide (the product of the enzymatic reaction) and further affected by protein-lipid interactions.Nowadays, considerable effort has therefore been made to substitute the aggregated POR complexes by recombinant monomeric enzymes, which are catalytically active. [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 Syn...

Photoinduced Self-Organization in “Semiconductor World”: Part I. from Protophotosynthesis to Protomembranes

“…In the subsequent evolutionary history of modern photosynthesis, this is recapitulated on a more complex biochemical base as an adaptation to natural light conditions in catalytic efficiency of photoenzymes. (It should be noted that these adaptation mechanisms aroused a discussion a few years ago [394][395][396] and some issues have not as yet been resolved, if we set aside discussions about model experiments that illustrate the arguments of the warring parties adaptation than proving anything).…”