Cytochrome f (Cyt f) is a structurally and evolutionarily unique c-type cytochrome whose physiological function it is to accept electrons from the Rieske iron-sulphur protein in the cytochrome bf complex and pass them on to small soluble redox proteins such as the cupredoxin plastocyanin in the thylakoid lumen. The interaction between Cyt f and its natural electron acceptors is highly transient; the need for specificity has to be balanced with the necessity for high turnover. The role of surface residues in determining this balance has been studied intensively in vitro (1-4). To increase our understanding of how in vitro experiments relate to crowded in vivo conditions, we have investigated the overall second order rate constant k2 of the interaction between plastocyanin and truncated, soluble Cyt f in the presence and absence of various low molecular weight viscogens or of macromolecular crowders, using stopped flow spectrophotometry. For small viscogens, the rate constant decreased with increasing viscosity of the buffer. No significant difference was observed between the viscogens ethane diol, glycerol or sucrose, despite the different molalities, volume occupancies and dielectric constants. The data suggest that two individual rate constants that contribute to k2 are diffusion limited. For the macromolecular crowders Dextran70 and Ficoll70, k2 did not change significantly with increasing viscosity. This suggests that the effects with small viscogens are associated with micro- rather than macroviscosity. However, at the concentration of crowders used, one would expect a rate enhancement caused by excluded volume effects, which might cancel out a viscosity effect.
References: 1. Schlarb-Ridley, B. G., Bendall, D. S., and Howe, C. J. (2002) Biochemistry 41, 3279-3285. 2. Schlarb-Ridley, B. G., Navarro, J. A., Spencer, M., Bendall, D. S., Hervas, M., Howe, C. J., and De La Rosa, M. A. (2002) Eur. J. Biochem. 269, 5893-5902. 3. Hart, S. E., Schlarb-Ridley, B. G., Delon, C., Bendall, D. S., and Howe, C. J. (2003) Biochemistry 42, 4829-4836. 4. Schlarb-Ridley, B. G., Bendall, D. S., and Howe, C. J. (2003) Biochemistry 42, 4057-4063.