Cytochrome-c₅₅₂ from thermus thermophilus: A functional and crystallographic investigation

The eubacterium Thermus thermophilus expresses terminal oxidases of the ba_3- and caa₃-type. The soluble cytochrome-c₅₅₂ of this organism has been isolated by a new method and characterized. In contrast to previous studies, but in line with coexpression at low aeration, the cytochrome was unambiguously identified as the substrate of the ba₃-oxidase. In the presence of TMPD and ascorbate, biphasic Eadie-Hofstee plots with k(max) = 250 s^-1 at 25°C are observed upon addition of cytochrome-c₅₅₂. Surprisingly, the caa₃-oxidase with its single covalently bound cytochrome-c also exhibits a biphasic redox activity with k(max) = 185 s^-1 in the presence of TMPD and ascorbate only. Further addition of cytochrome-c₅₅₂ does not lead to enhanced activity. Crystals of cytochrome-c₅₅₂ were obtained by vapor diffusion using the sitting-drop method in the presence of ammonium sulfate as precipitant. They diffract to 1.28 Å resolution using synchrotron radiation. The structure has been solved by MAD phasing.