Cofactor-dependent structural and binding properties of yeast cytochrome C peroxidase

Yeast cytochrome c peroxidase (CcP) is a heme enzyme that reduces hydroperoxides using the electrons provided by its physiological partner cytochrome c (Cc). Contributing to the resistance against the oxidative stress associated with the aerobic metabolism, the Cc-CcP complex has been widely studied and became a paradigm for biological electron transfer. The heme-free, enzymatically inactive apo CcP is the natural precursor of the mature, cofactor-bound holo protein. Despite its physiological relevance, apo CcP is not well characterized, and at present, little is known about its structure or the interaction with Cc. Using a range of biophysical techniques, here we show that, while holo CcP binds Cc with micromolar affinity, the interaction between apo CcP and Cc is completely abolished. Characterized by small-angle X-ray scattering, solution nuclear magnetic resonance spectroscopy, and equilibrium unfolding experiments, apo and holo CcP exhibit very similar structural, hydrodynamic, and thermodynamic properties. However, detailed analysis reveals that apo CcP is more expanded in solution, displays a number of characteristics associated with a molten globule state, and, unlike the holo protein, does not form an unfolding intermediate during thermal and chemical denaturation. Overall, our data suggest that the Cc binding site present in the holo protein is disrupted in the apo form, explaining the inability of the latter to interact with Cc. We argue that the observed difference in Cc binding is physiologically relevant and suggest why abolishing the apo CcP-Cc interaction is beneficial to the organism.