Expression, purification and labeling of recombinant horse cytochrome c, and measurements of photoinduced intraprotein electron transfer

Katalin Tenger, Gábor Rákhely, Petro Khoroshyy and László Zimányi

Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary

1-thiouredopyrene-3,6,8-trisulfonate (TUPS) has recently been used as a photoinduced covalent redox label capable of reducing various cofactors of proteins. Single cysteine residues were introduced on the surface of horse cytochrome c for covalent labeling by TUPS. The protein was heterologously expressed in E. coli together with yeast heme lyase to assist heme binding to the cytochrome. In former studies a chemically different version of TUPS was used to label lysine side chains, and the kinetics of forward and reverse electron transfer between TUPS and the heme were measured. Here we report the comparison of electron transfer rates when some of these lysines are replaced by cysteine, thereby altering both the chemical nature and the length of the link between the electron donor-acceptor pair.

Email: tenger@nucleus.szbk.u-szeged.hu

Address: Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, P.O. BOX 521, 6701 Szeged, Hungary