Cross-Linking between Cytochromec3and Flavodoxin fromDesulfovibrio gigas

Abstract

Tetraheme cytochromec3(13 kDa) and flavodoxin (16 kDa), are small electron transfer proteins that have been used to mimic,in vitro,part of the electron-transfer chain that operates between substract electron donors and respiratory electron acceptors partners inDesulfovibriospecies (Palma, N., Moura, I., LeGall, J., Van Beeumen, J., Wampler, J., Moura, J. J. G. (1994)Biochemistry33, 6394–6407). The electron transfer between these two proteins is believed to occur through the formation of a specific complex where electrostatic interaction is the main driving force (Stewart, D., LeGall, J., Moura, I., Moura, J.J.G., Peck, H.D., Xavier, A.V., Weiner, P.K. and Wampler, J.E. (1988)Biochemistry27, 2444–2450, Stewart, D., LeGall, J., Moura, I., Moura, J.J.G., Peck, H.D., Xavier, A.V., Weiner, P., Wampler, J. (1989)Eur. J. Biochem.185, 695–700). In order to obtain structural information of the pre-complex, a covalent complex between the two proteins was prepared. A water-soluble carbodiimide [EDC (1-ethyl-3(3 dimethylaminopropyl) carbodiimide hydrochloride] was used for the cross linking reaction. The reaction was optimized varying a wide number of experimental parameters such as ionic strength, protein and cross linker concentration, and utilization of different cross linkers and reaction time between the crosslinker and proteins.