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We have succeeded in preparing a mutant of the High Potential Iron−Sulfur Protein (HiPIP) from Chromatium vinosum in which a cysteine ligand has been replaced by a serine (C77S). Proton chemical shift data and nuclear Overhauser effects indicate that structural perturbations induced by the C77S mutation are minimal in both the oxidized and reduced forms of the HiPIP. The reduction potential of C77S is 25 mV lower than that of the wild-type HiPIP (WT) (0.2 M ionic strength, pH 4.5−9.0, 25 °C). Assignment of the hyperfine shifted signals in the ¹H NMR spectrum of oxidized C77S revealed that the temperature dependences of the signals associated with cluster-ligating residues 46 and 77 are Curie and anti-Curie type, respectively, and are thus the reverse of those in WT. Taken together, these observations indicate that the iron bound to Ser-77 is less reducible than the corresponding iron in WT. The results are …