Cross-Linking between Cytochrome c3 and Flavodoxin from Desulfovibrio gigas

Tetraheme cytochrome c3 (13 kDa) and flavodoxin
(16 kDa), are small electron transfer proteins that
have been used to mimic, in vitro, part of the electrontransfer
chain that operates between substract electron
donors and respiratory electron acceptors partners
in Desulfovibrio species (Palma, N., Moura, I.,
LeGall, J., Van Beeumen, J., Wampler, J., Moura,
J. J. G. (1994) Biochemistry 33, 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) Biochemistry 27, 2444–2450, Stewart, D., Le-
Gall, 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.