Hydrogen exchange behavior of [U-N-15]-Labeled oxidized and reduced iso-1-cytochrome c

Heteronuclear NMR spectroscopy was used to measure the hydrogen-deuterium e xchange rates of backbone amide hydrogens in both oxidized and reduced [U-N -15]iso-1-cytochrome c from the yeast Saccharomyces cerevisiae. The exchang e data confirm previously reported data [Marmorino et al. (1993) Protein Sc i. 2, 1966-1974], resolve several inconsistencies, and provide more thoroug h coverage of exchange rates throughout the cytochrome c protein in both ox idation states. Combining the data previously collected on unlabeled C102T with the current data collected on [U-15N]C102T, exchange rates for 53 prot ons in the oxidized state and 52 protons in the reduced state can now be re ported. Most significantly, hydrogen exchange measurements on [U-N-15]iso-1 -cytochrome c allowed the observation of exchange behavior of the secondary structures, such as large loops, that are not extensively hydrogen-bonded. For the helices, the most slowly exchanging protons are found in the middl e of the helix, with more rapidly exchanging protons at the helix ends. The observation for the Omega-loops in cytochrome c is just the opposite. In t he loops, the ends contain the most slowly exchanging protons and the loop middles allow more rapid exchange. This is found to be true in cytochrome c loops, even though the loop ends are not attached to any regular secondary structures. Some of the exchange data are strikingly inconsistent with dat a collected on the C102S variant at a different pH, which suggests pH-depen dent dynamic differences in the protein structure. This new hydrogen exchan ge data for loop residues could have implications for the substructure mode l of eukaryotic cytochrome c folding. Isotopic labeling of variant forms of cytochrome c can now be used to answer many questions about the structure and folding of this model protein.