A solution model of the complex formed by adrenodoxin and adrenodoxin reductase determined by paramagnetic NMR spectroscopy.

Lanthanide tags offer the opportunity to retrieve long-range distance information from NMR experiments that can be used to guide protein docking. To determine whether sufficient restraints can be retrieved for proteins with low solubility and availability, Ln tags were applied in the study of the 65 kDa membrane-associated protein complex formed by the electron carrier adrenodoxin and its electron donor, adrenodoxin reductase. The reductase is only monomeric at low concentration, and the paramagnetic iron-sulfur cluster of adrenodoxin broadens many of the resonances of nuclei in the interface. Guided by the paramagnetic restraints obtained using two Ln-tag attachment sites, protein docking yields a cluster of solutions with an rmsd of 3.2 A. The mean structure is close to the crystal structure of the cross-linked complex, with an rmsd of 4.0 A. It is concluded that with the application of Ln tags paramagnetic NMR restraints for structure determination can be retrieved even for difficult, low-concentration protein complexes.

Intermolecular dynamics studied by paramagnetic tagging.

Yeast cytochrome c and bovine adrenodoxin form a dynamic electron transfer complex, which is a pure encounter complex. It is demonstrated that the dynamic nature of the interaction can readily be probed by using a rigid lanthanide tag attached to cytochrome c. The tag, Caged Lanthanide NMR Probe 5, induces pseudocontact shifts and residual dipolar couplings and does not perturb the binding interface. Due to the dynamics in the complex, residual dipolar couplings in adrenodoxin are very small. Simulation shows that cytochrome c needs to sample a large part of the surface of adrenodoxin to explain the small degree of alignment observed for adrenodoxin. The applied method provides a simple and straightforward way to observe dynamics in protein complexes or domain-domain mobility without the need for external alignment media.

Dynamics in a pure encounter complex of two proteins studied by solution scattering and paramagnetic NMR spectroscopy.

In the general view of protein-complex formation, a transient and dynamic encounter complex proceeds to form a more stable, well-defined, and active form. In weak protein complexes, however, the encounter state can represent a significant population of the complex. The redox proteins adrenodoxin (Adx) and cytochrome c (C c) associate to form such a weak and short-lived complex, which is nevertheless active in electron transfer. To study the conformational freedom within the protein complex, the native complex has been compared to a cross-linked counterpart by using solution scattering and NMR spectroscopy. Oligomerization behavior of the native complex in solution revealed by small-angle X-ray scattering indicates a stochastic nature of complex formation. For the cross-linked complex, interprotein paramagnetic effects are observed, whereas for the native complex, extensive averaging occurs, consistent with multiple orientations of the proteins within the complex. Simulations show that C c samples about half of the surface area of adrenodoxin. It is concluded that the complex of Adx/C c is entirely dynamic and can be considered as a pure encounter complex.

The adrenodoxin-like ferredoxin of Schizosaccharomyces pombe mitochondria.

The single mitochondrial type I [2Fe-2S] ferredoxin of the fission yeast Schizosaccharomyces pombe is produced as the carboxy terminal part of the electron-transfer-protein 1 (etp1) and cleaved off during mitochondrial import [Biochemistry 41 (2002) 2311-2321]. The UV/Vis (UV-visible) spectrum of the purified recombinant ferredoxin domain (etp1(fd)) expressed in Escherichia coli is similar to those of bovine Adx in the oxidized as well as in the reduced state. EPR (electronic paramagnetic resonance) studies revealed a correctly incorporated iron-sulfur cluster of the axial type. The redox potential of this protein was determined to be -353 mV, which is considerably lower than that of adrenodoxin (Adx, -273 mV). Several lines of evidence indicate that the protein forms dimers under physiological and denaturating conditions. Interestingly, the fission yeast ferredoxin could be shown to be active as an electron carrier in heterologous redox systems. It is able to transfer electrons to horse heart cytochrome c and to bovine cytochromes P450(scc) (CYP11A1) and P450(11 beta) (CYP11B1), thereby receiving electrons from bovine NADPH-dependent Adx reductase. The kinetics of substrate conversion in the etp1(fd)-supported CYP11A1 and CYP11B1-dependent systems mediated was studied.

Transient protein interactions studied by NMR spectroscopy: the case of cytochrome C and adrenodoxin.

The interaction between yeast iso-1-cytochrome c (C102T) and two forms of bovine adrenodoxin, the wild type and a truncated form comprising residues 4-108, has been investigated using a combination of one- and two-dimensional heteronuclear NMR spectroscopy. Chemical shift perturbations and line broadening of amide resonances in the [(15)N,(1)H]HSQC spectrum for both (15)N-labeled cytochrome c and adrenodoxin in the presence of the unlabeled partner protein indicate the formation of a transient complex, with a K(a) of (4 +/- 1) x 10(4) M(-)(1) and a lifetime of <3 ms. The perturbed residues map over a large surface area for both proteins. For cytochrome c, the dominating effects are located around the exposed heme edge but with other areas also affected upon formation of the complex. In the case of adrenodoxin, effects are seen in both the recognition and core domains, with the largest perturbations in the recognition domain. These results indicate that the complex has a dynamic nature, with delocalized binding of cytochrome c on adrenodoxin. A comparison with other transient complexes of redox proteins places this complex between well-defined complexes such as the cytochrome c-cytochrome c peroxidase complex and entirely dynamic complexes such as the cytochrome b(5)-myoglobin complex.