Macromolecular crowding refers to the phenomenon in which biomolecular properties are altered due to high concentrations of macromolecules in an environment. In order to examine the putative effects of macromolecular crowding on biological redox reactions, the iron-sulfur protein ferredoxin (Fdx) and the FAD-containing flavoprotein ferredoxin-NADP+ reductase (FNR) from spinach were selected as a model system. Purification of the proteins from Spinacea oleracea was implemented through tissue homogenization and sonication followed by a series of chromatographic steps. With NADPH as a reductant, FNR is capable of reducing the dye dichlorophenolindophenol (DCIP) directly, and is also capable of reducing mammalian cytochrome c in a Fdx-dependent reaction. Steady state kinetics of cytochrome c reduction and DCIP reduction were measured using an NADPH-generating system. The polymers Ficoll and Dextran were utilized as model macromolecular crowding species at concentrations ranging up to 8%. Ovalbumin as a model protein crowding agent was also examined at concentrations up to 4%. Whereas the molecular crowding agents showed mixed effects on the rate of DCIP reduction, significant inhibitory effects were observed for the rates of ferredoxin-dependent cytochrome c reduction kinetics. Extent of inhibition was also dependent on the molecular weight of the crowding species.

Investigation of macromolecular crowding on ferredoxin and ferredoxin-NADP+ reductase kinetics

249th National Meeting (2015)

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