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 ...

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Thrombocytopenia 4 is an inherited autosomal dominant thrombocytopenia, which occurs due to mutations in the human gene for cytochrome c that results in enhanced mitochondrial apoptotic activity. The Gly41Ser mutation was the first to be reported. Here we report stopped-flow kinetic studies of azide binding to human ferricytochrome c and its Gly41Ser variant, together with backbone amide H/D exchange and 15N-relaxation dynamics using NMR spectroscopy, to show that alternative conformations are kinetically and thermodynamically more readily accessible for the Gly41Ser variant than for the wild-type protein. Our work reveals a direct conformational link between the 40–57 Ω-loop in which residue 41 resides and the dynamical properties of the axial ligand to the heme iron, Met80, such that the replacement of glycine by serine promotes the dissociation of the Met80 ligand, thereby increasing the population of a peroxidase active state, which is a key non-native conformational state in apoptosis.

We have investigated whether the pro-apoptotic properties of the G41S mutant of human cytochrome c can be explained by a higher than wild-type peroxidase activity triggered by phospholipid binding. A key complex in mitochondrial apoptosis involves cytochrome c and the phospholipid cardiolipin. In this complex cytochrome c has its native axial Met(80) ligand dissociated from the haem-iron, considerably augmenting the peroxidase capability of the haem group upon H2O2 binding. By EPR spectroscopy we reveal that the magnitude of changes in the paramagnetic haem states, as well as the yield of protein-bound free radical, is dependent on the phospholipid used and is considerably greater in the G41S mutant. A high-resolution X-ray crystal structure of human cytochrome c was determined and, in combination with the radical EPR signal analysis, two tyrosine residues, Tyr(46) and Tyr(48), have been rationalized to be putative radical sites. Subsequent single and double tyrosine-to-phenylalanine mutations revealed that the EPR signal of the radical, found to be similar in all variants, including G41S and wild-type, originates not from a single tyrosine residue, but is instead a superimposition of multiple EPR signals from different radical sites. We propose a mechanism of multiple radical formations in the cytochrome c-phospholipid complexes under H2O2 treatment, consistent with the stabilization of the radical in the G41S mutant, which elicits a greater peroxidase activity from cytochrome c and thus has implications in mitochondrial apoptosis.

The interaction of yeast iso-1-cytochrome c with its physiological redox partner cytochrome c peroxidase has been investigated using heteronuclear NMR techniques. Chemical shift perturbations for both 15N and 1H nuclei arising from the interaction of isotopically enriched 15N cytochrome c with cytochrome c peroxidase have been observed. For the diamagnetic, ferrous cytochrome c, 34 amides are affected by binding, corresponding to residues at the front face of the protein and in agreement with the interface observed in the 1:1 crystal structure of the complex. In contrast, for the paramagnetic, ferric protein, 56 amides are affected, corresponding to residues both at the front and toward the rear of the protein. In addition, the chemical shift perturbations were larger for the ferric protein. Using experimentally observed pseudocontact shifts the magnetic susceptibility tensor of yeast iso-1-cytochrome c in both the free and bound forms has been calculated with HN nuclei as inputs. In contrast to an earlier study, the results indicate that there is no change in the geometry of the magnetic axes for cytochrome c upon binding to cytochrome c peroxidase. This leads us to conclude that the additional effects observed for the ferric protein arise either from a difference in binding mode or from the more flexible overall structure causing a transmittance effect upon binding.