Solution structure and dynamics of the complex between cytochromecand cytochromecperoxidase determined by paramagnetic NMR

Abstract

The physiological complex of yeast cytochrome c peroxidase and iso-1-cytochrome c is a paradigm for biological electron transfer. Using paramagnetic NMR spectroscopy, we have determined the conformation of the protein complex in solution, which is shown to be very similar to that observed in the crystal structure [ electron transfer ͉ encounter state ͉ transient complex ͉ spin label ͉ paramagnetic relaxation enhancement T he process of protein complex formation can be described by a two-step model, in which a short-lived, dynamic encounter complex precedes a dominant, well defined state (Fig. 1). The former enables proteins to undergo reduced-dimensionality search of the optimal binding geometry, thereby accelerating molecular association as compared with 3D diffusion (1). Fast molecular association is essential for protein-protein complexes that require high turnover rates, like those involved in electron transfer (ET) in photosynthesis, respiration, and other metabolic processes (2). The physiological complex of yeast iso-1-cytochrome c (Cc) and yeast cytochrome c peroxidase (CcP) is a paradigm for the intermolecular ET (3) and is one of the few transient ET complexes for which a crystal structure has been solved (4). A recent study has confirmed that the protein-protein orientation observed in the crystal is ET-active (5). However, it has remained a matter of debate whether this structure represents the only form in solution (6-8). According to several studies, the complex is dynamic, and the crystal structure might represent only a subpopulation of protein orientations (9-15). Recent studies show that the photoinduced ET between Znsubstituted CcP and Cc, both in the crystal (8, 16) and in solution (17), occurs with faster backward than forward rates, indicating that the complex is present in multiple forms, only a few of which are .Characterization of the binding interface in the dynamic encounter state (Fig. 1B) has so far proven to be elusive. Investigation of protein complexes by using x-ray crystallography or conventional NMR spectroscopy addresses only the singleorientation species (Fig. 1C), and the only way to visualize the dynamic state is offered by theoretical modeling studies (11,18). In the recent, elegant work of Clore and coworkers (19,20), it was shown how paramagnetic relaxation can be applied to study the dynamic state of protein-DNA complexes. We report on the application of an analogous experimental approach that allows us to define both the dominant protein-protein orientation (Fig. 1C) and the conformational space sampled by the proteins in the dynamic encounter complex (Fig. 1B). Results and DiscussionSolution Structure of the Complex. The concept of the approach is that NMR resonance intensities of one of the proteins in the complex are affected by a paramagnetic spin label covalently attached to the other protein (Fig. 2). The paramagnetic effects are converted into distance restraints (21-23), which can be used to calculate protein-protein orientations within the complex (24). Five ...

Effect of Single-Site Charge-Reversal Mutations on the Catalytic Properties of Yeast Cytochrome c Peroxidase: Mutations near the High-Affinity Cytochrome c Binding Site

“…The encounter complexes encompass much of the CcP surface as first postulated by Northrup et al (14). Using covalently attached paramagnetic spin labels, Volkov et al (31) show that encounter complexes exist near residues 38, 200, and 288 on the surface of CcP and but not at residues 137 and 263. The spin label probe at residue 38 should detect cytochrome c binding near Asp-37.…”

“…The former mutation causes a 30-fold decrease in binding affinity while the latter has essentially no effect. Assuming that the D37K mutation causes a localized effect on cytochrome c binding leads to the conclusion that cytochrome c can bind in a different orientation from that shown in the crystal structure of the 1:1 complex, one that involves direct interaction between Asp-37 and a positively charged residue on cytochrome c. Alternatively, Asp-37 could be part of a second binding site that is adjacent to and/or overlaps the crystallographic site as suggested by Nocek et A recent NMR study (31) of the CcP/yeast iso-1 cytochrome c complex demonstrates that the bound cytochrome has considerable mobility, but generally corroborates the crystallographic structure, indicating that cytochrome c resides at the crystallographic site about 70% of the time and in much more dynamic encounter complexes about 30% of the time. The encounter complexes encompass much of the CcP surface as first postulated by Northrup et al (14).…”

“…Since that time a large number of studies have been published concerning the nature of the cytochrome c/CcP interaction and the potential locations of the cytochrome c binding sites on CcP (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). Northrup et al published an influential study modeling the electrostatic interaction between the positively charged cytochrome c molecule and the negatively charged CcP using Brownian dynamics simulations (14).…”

Mechanistic details of a protein–protein association pathway revealed by paramagnetic relaxation enhancement titration measurements

“…Despite the importance of encounter complex ensembles in protein-protein association, little is known of their structures and configurations because their populations are low, their lifetimes are short, and they are difficult to trap, rendering them essentially invisible to conventional structural and biophysical methods. Recently, however, the application of NMR paramagnetic relaxation enhancement (PRE), a technique that is exquisitely sensitive to the presence of lowly populated states in the fast exchange regime (12,13), has offered new insights into the physicochemical and structural nature of transient encounter complexes in protein-DNA (14) and protein-protein (15)(16)(17)(18)(19)(20) association. The mechanism, however, whereby the interacting molecules in the encounter complex ensemble find their final stereospecific structure, remains poorly understood.…”

Direct Visualization Reveals Dynamics of a Transient Intermediate During Protein Assembly

“…Experimental studies of transient intermediates are still at the infant stage, because these intermediates have short lifetimes and are rarely populated at equilibrium. Pioneering NMR studies have revealed the structures of rare conformational states in equilibrium with the predominant structure in the apo-protein or the final complex, and provided direct experimental support for the ability of proteins to explore different conformations (13)(14)(15)(16)(17). Nevertheless, many of these studies have focused on protein interactions that are inherently weak and nonspecific; whether the same principle applies to the assembly of a stable and stereospecific protein complex remains to be determined.…”