STRUCTURE AND DYNAMICS OF CYTOCHROME-C IN NONAQUEOUS SOLVENTS BY 2D NH-EXCHANGE NMR-SPECTROSCOPY

Recent biocatalysis studies have shown that many enzymes exhibit catalytic activity and enhanced thermal stability in essentially anhydrous nonpolar solvents. At present we do not understand the important role that water plays in protein structure stabilization and in enzyme mechanisms in nonaqueous environments. In this study, the NH-exchange rates of oxidized horse cytochrome c suspended in tetrahydrofuran (THF) containing 1% D2O (vol/vol) at 37-degrees-C, pH 8.9, have been determined indirectly by 2D NH-exchange NMR spectroscopy. In such a solvent system, we have allowed just enough water molecules to form approximately a monolayer surrounding the protein. A hydrophobic tripeptide N-acetyl-(Ala)3-OCH3 was used to calculate the intrinsic amide-exchange rate directly for a random-coil peptide in THF/1% D2O. The relative solvent protection factors for amide exchange of cytochrome c in THF/1% D2O have been determined for 35 amide protons and compared with the results obtained at the same pH in aqueous solution. Almost all of these protected protons are located in the three major alpha-helical regions or involved in crystallographically defined intramolecular hydrogen bonding. In both aqueous solution and THF/1% D2O at pH 8.9, the NH-exchange profile for cytochrome c is similar to that in the native state (pD 7.0: Jeng, et al., Biochemistry 1990, 29, 10433-10437). The protection factors against exchange were greatest for the NH protons of Phe10 and those in the central part of the C-terminal helix, Ile95, Tyr97, and Leu98. The protection factors from the majority of observable NH protons for cytochrome c in purely aqueous solution, pH 8.9, vary between 10(4) and 10(8). These are about <50-fold smaller than those in the native state at pD 7.0 but are 10(3)-10(4) larger than those in the molten globule state. The protection factors in THF/1 % D2O at pH 8.9 vary between 10(2) and 10(5), which are in turn 10(2)-10(3)-fold smaller than those observed at pH 8.9 in purely aqueous solution. These results suggest that while cytochrome c remains folded and compact at pH 8.9 in both purely aqueous solution and THF/1% D2O, the flexibility of the protein is clearly enhanced, especially in the organic medium. However, some differences were also observed between the two solvent systems. Most noticeably part of the C-terminal helix (Leu94, Ala96, and Lys99) has the lowest NH-exchange rates in D2O, whereas in THF/1% D2O these exchange rates increased dramatically. This may reflect differences in tertiary interactions in the two solvents. The N-terminal and C-terminal helices fold over each other in the native structure. The decrease in protection factor for those residues of the C-terminal helix facing the N-terminal helix suggests that this helix interaction is destabilized in the hydrated organic solvent, exposing to the surface residues Leu94, Ala96, and Lys99. These hydrogen-exchange results on cytochrome c suggest that water serves to ''lubricate'' the local dynamics of the protein in THF/1% D2O and that the slightly hydrated organic solvent enhances the dynamic flexibility of all regions of the native structure.