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 Miyazaki F have been located in the electron-density map calculated at 4 A 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 successfully assigned to three iron-sulfur clusters and one Ni atom by a difference Fourier technique with coefficients of the best phases from the multiple isomorphous replacement (MIR) method and structure factors obtained at five wavelengths (1.040, 1.487, 1.730, 1.743 and 1.750 A) with the use of a synchrotron radiation source. Four active centers are approximately lined up at a distance of ca 13 A, which seems reasonable if they are connected with the electron-transfer chain.
Department of Life Sciences, Himeji Institute of Technology, Hyogo, Japan.
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Higuchi
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Okamoto
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Fujimoto
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Misaki