Structure and Function of Transient Encounters of Redox Proteins

Many biomolecular interactions proceed via lowly populated, transient intermediates. Believed to facilitate formation of a productive complex, these short-lived species are inaccessible to conventional biophysical and structural techniques and, until recently, could only be studied by theoretical simulations. Recent development of experimental approaches sensitive to the presence of minor species--in particular paramagnetic relaxation enhancement (PRE) NMR spectroscopy--has enabled direct visualization and detailed characterization of such lowly populated states. Collectively referred to as an encounter complex, the binding intermediates are particularly important in transient protein interactions, such as those orchestrating signaling cascades or energy-generating electron transfer (ET) chains. Here I discuss encounter complexes of redox proteins mediating biological ET reactions, which are essential for many vital cellular activities including oxidative phosphorylation and photosynthesis. In particular, this Account focuses on the complex of cytochrome c (Cc) and cytochrome c peroxidase (CcP), which is a paradigm of biomolecular ET and an attractive system for studying protein binding and enzymatic catalysis. The Cc-CcP complex formation proceeds via an encounter state, consisting of multiple protein-protein orientations sampled in the search of the dominant, functionally active bound form and exhibiting a broad spatial distribution, in striking agreement with earlier theoretical simulations. At low ionic strength, CcP binds another Cc molecule to form a weak ternary complex, initially inferred from kinetics experiments and postulated to account for the measured ET activity. Despite strenuous efforts, the ternary complex could not be observed directly and remained eagerly sought for the past two decades. Very recently, we have solved its structure in solution and shown that it consists of two binding forms: the dominant, ET-inactive geometry and an ensemble of lowly populated species with short separations between Cc and CcP cofactors, which summarily account for the measured ET rate. Unlike most protein complexes, which require accurate alignment of the binding surfaces in a single, well-defined orientation to carry out their function, redox proteins can form multiple productive complexes. As fast ET will occur any time the redox centers of the binding partners are close enough to ensure efficient electron tunneling across the interface, many protein-protein orientations are expected to be ET active. The present analysis confirms that the low-occupancy states can support the functional ET activity and contribute to the stability of redox protein complexes. As illustrated here, boundaries between the dominant and the encounter forms become blurred for many dynamic ET systems, which are more aptly described by ensembles of functionally and structurally heterogeneous bound forms.