Protein hydration and location of water molecules in oxidized horse heart cytochrome c by H-1 NMR

The hydration properties of the oxidized form of horse heart cytochrome c h ave been studied by H-1 NMR spectroscopy. Two-dimensional, homonuclear ePHO GSY-NOESY experiments are used to map water-protein interactions. The detec ted NOEs reveal interactions between nonexchangeable protein protons and bo th water protons and labile protein protons which exchange with water proto ns. Among the many water molecules apparent in the X-ray structure, three h ave been identified with a residence time longer than 300 ps. One of them i s located inside the distal heme cavity, in the deepest part of a hydration pathway extending toward the surface. The identification of hydrophilic re gions and detection of three long-lived water molecules settles some ambigu ities and provides a better representation of the water-protein interaction s in oxidized cytochrome c. (C) 2000 Academic Press.