COMPLEX OF PLASTOCYANIN AND CYTOCHROME-C CHARACTERIZED BY NMR CHEMICAL-SHIFT ANALYSIS

The complexes of horse ferrous and ferric cytochrome c with Cd-substit uted pea plastocyanin have been characterized by nuclear magnetic reso nance, in order to determine the binding sites and to study the effect s of complex formation. Reproducible, small chemical shift changes (0. 005-0.05 ppm) were observed for protons in both proteins upon formatio n of a 1:1 complex. The chemical shift changes depended on the ratio o f free to bound protein, with a binding constant of 1.0 +/- 0.5 x 10(5 ) M-1, indicating that they were caused by complex formation and that free and bound proteins were in fast exchange. Two-dimensional spectra of the complex of ferrocytochrome c and plastocyanin were screened sy stematically for chemical shift changes. For about 760 protons, or 70% of the assigned protons in the two proteins, the chemical shift in th e complex could be established. In plastocyanin and cytochrome c 14% a nd 17% of the protons, respectively, showed a significant chemical shi ft change. These protons form two groups. The first consists of a limi ted number of surface-exposed side-chain protons. These map on the so- called east side of plastocyanin and the front side of cytochrome c. T his group of chemical shift changes is interpreted as representing dir ect effects of binding, and the respective surfaces thus represent the binding sites. The second group includes backbone amide protons and a few aliphatic and aromatic protons in the hydrophobic core of each pr otein. The chemical shift changes of this group are interpreted as sec ondary, i.e., caused by very small structural changes which are transm itted deep into the core of the protein. Ferric cytochrome c caused th e same chemical shift effects in plastocyanin as the ferrous form; no intermolecular paramagnetic effects were observed. The small size of t he chemical shifts and the absence of intermolecular paramagnetic shif ts and NOEs suggest that the complex consists of a dynamic ensemble of structures which are in fast exchange, rather than a single static co mplex. This study shows that small, reproducible chemical shifts can b e used effectively to characterize protein complexes in detail.