Complex of plastocyanin and cytochrome c characterized by NMR chemical shift analysis.

Abstract

The complexes of horse ferrous and ferric cytochrome c with Cd-substituted pea plastocyanin have been characterized by nuclear magnetic resonance, in order to determine the binding sites and to study the effects of complex formation. Reproducible, small chemical shift changes (0.005-0.05 ppm) were observed for protons in both proteins upon formation of a 1:1 complex. The chemical shift changes depended on the ratio of 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 systematically for chemical shift changes. For about 760 protons, or 70% of the assigned protons in the two proteins, the chemical shift in the complex could be established. In plastocyanin and cytochrome c 14% and 17% of the protons, respectively, showed a significant chemical shift change. These protons form two groups. The first consists of a limited number of surface-exposed side-chain protons. These map on the so-called east side of plastocyanin and the front side of cytochrome c. This group of chemical shift changes is interpreted as representing direct 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 protein. The chemical shift changes of this group are interpreted as secondary, i.e., caused by very small structural changes which are transmitted deep into the core of the protein. Ferric cytochrome c caused the same chemical shift effects in plastocyanin as the ferrous form; no intermolecular paramagnetic effects were observed. The small size of the chemical shifts and the absence of intermolecular paramagnetic shifts and NOEs suggest that the complex consists of a dynamic ensemble of structures which are in fast exchange, rather than a single static complex. This study shows that small, reproducible chemical shifts can be used effectively to characterize protein complexes in detail.