Metalloprotein complexes for the study of electron-transfer reactions. Characterization of diprotein complexes obtained by covalent cross-linking of cytochrome c and plastocyanin with a carbodiimide.
Cytochrome c (cyt) and zinc cytochrome c (Zncyt) are separately cross-linked to plastocyanin (pc) by the carbodiimide EDC according to a published method. The changes in the protein reduction potentials indicate the presence of approximately two amide cross-links. Chromatography of the diprotein complexes cyt/pc and Zncyt/pc on CM-52 resin yields multiple fractions, whose numbers depend on the eluent. UV-vis, EPR, CD, MCD, resonance Raman, and surface-enhanced resonance Raman spectra show that cross-linking does not significantly perturb the heme and blue copper active sites. Degrees of heme exposure show that plastocyanin covers most of the accessible heme edge in cytochrome c. Impossibility of cross-linking cytochrome c to a plastocyanin derivative whose acidic patch had been blocked by chemical modification shows that it is the acidic patch that abuts the heme edge in the covalent complex. The chromatographic fractions of the covalent diprotein complex are structurally similar to one another and to the electrostatic diprotein complex. Isoelectric points show that the fractions differ from one another in the number and distribution of N-acylurea groups, byproducts of the reaction with the carbodiimide. Cytochrome c and plastocyanin are also tethered to each other via lysine residues by N-hydroxysuccinimide diesters. Tethers, unlike direct amide bonds, allow mobility of the cross-linked molecules. Laser-flash-photolysis experiments show that, nonetheless, the intracomplex electron-transfer reaction cyt(II)/pc(II)----cyt(III)/pc(I) is undetectable in complexes of either type. Only the electrostatic diprotein complex, in which protein rearrangement from the docking configuration to the reactive configuration is unrestricted, undergoes this intracomplex reaction at a measurable rate.
Zhou
J S
JS
Department of Chemistry, Iowa State University, Ames 50011.
Brothers
H M
HM
Neddersen
J P
JP
Peerey
L M
LM
Cotton
T M
TM
Kostić
N M
NM
D000577:Amides / Q000737:chemistry; D002852:Chromatography, Ion Exchange; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003574:Cytochrome c Group / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry*; D007700:Kinetics; D008667:Metalloproteins / Q000737:chemistry* / Q000302:isolation & purification; D010084:Oxidation-Reduction; D010970:Plastocyanin / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry
D000577:Amides; D003432:Cross-Linking Reagents; D003574:Cytochrome c Group; D008667:Metalloproteins; D010970:Plastocyanin; D015032:Zinc; D005022:Ethyldimethylaminopropyl Carbodiimide