IMPORTANCE OF PROTEIN REARRANGEMENT IN THE ELECTRON-TRANSFER REACTION BETWEEN THE PHYSIOLOGICAL PARTNERS CYTOCHROME-F AND PLASTOCYANIN

Cytochrome f from turnip and plastocyanin from French bean were noninvasively cross-linked in the presence of the carbodiimide EDC so that the exposed heme edge in the former protein abuts the acidic patch remote from the copper site in the latter [Morand, L. Z., Frame, M. K., Colvert, K. K., Johnson, D. A., Krogmann, D. W., & Davis, D. J. (1989) Biochemistry 28, 8039]. The molecular mass, reduction potentials, and UV-visible and ESR spectra of the covalent complex were consistent with the composition cyt/pc and with a lack of noticeable structural perturbations of the protein molecules. Isoelectric focusing showed the presence of N-acylurea groups, byproducts of the cross-linking reaction [Zhou, J. S., Brothers, H. M. II, Neddersen, J. P., Peerey, L. M., Cotton, T. M., & Kostic, N. M. (1992) Bioconjugate Chem. 3, 382]. Laser flash spectroscopy, with riboflavin semiquinone as the reductant, showed that the electron-transfer reaction within the covalent complex cyt(II)//pc(II) is either undetectably slow or reversible. The question was resolved by monitoring, during redox titrations, the H-1 NMR line widths of the heme methyl groups in free ferricytochrome f and in this protein cross-linked to plastocyanin. Line broadening showed that the intracomplex reaction does occur in the electrostatic complex cyt/pc; the lower limit of the rate constant, estimated from the line broadening, was consistent with the actual value of 2800 +/- 300 s-1, determined previously [Qin, L., & Kostic, N. M. (1992) Biochemistry 31, 5145]. These experiments verified the applicability of H-1 NMR spectroscopy to the study of electron-transfer reactions between cytochrome f and plastocyanin. Absence of noticeable line broadening during redox titrations of the covalent complex cyt/pc showed that the intracomplex electron-transfer reaction is undetectably slow; the upper limit is ca. 0.1 s-1. Control experiments by stopped-flow spectrophotometry showed that N-acylurea groups do not appreciably alter the intrinsic electron-transfer reactivity of the proteins. Therefore it is the cross-links that abolish the intracomplex electron-transfer reaction in the covalent complex. Recent studies by others indicated that, in the noncovalent complex, ferrocytochrome f reduces cupriplastocyanin from the acidic patch. The present study indicates that this reaction between physiological partners requires conformational fluctuations of the noncovalent diprotein complex, which are impeded by cross-links.