LOCATION OF ACTIVE-SITES OF NIFE HYDROGENASE DETERMINED BY THE COMBINATION OF MULTIPLE ISOMORPHOUS REPLACEMENT AND MULTIWAVELENGTH ANOMALOUS-DIFFRACTION METHODS

The active centers of NiFe hydrogenase from Desulfovibrio vulgaris Miy azaki F have been located in the electron-density map calculated at 4 angstrom resolution. The electron-density map based on five heavy-atom derivatives showed four strong peaks which were clearly distinguished from the protein region. These strong densities have been successfull y assigned to three iron-sulfur clusters and one Ni atom by a differen ce Fourier technique with coefficients of the best phases from the mul tiple isomorphous replacement (MIR) method and structure factors obtai ned at five wavelengths (1.040, 1.487, 1.730, 1.743 and 1.750 angstrom ) with the use of a synchrotron radiation source. Four active centers are approximately lined up at a distance of ca 13 angstrom, which seem s reasonable if they are connected with the electron-transfer chain.