Cytochrome-c552 from Thermus thermophilus: a functional and crystallographic investigation.

The eubacterium Thermus thermophilus expresses terminal oxidases of the ba3- 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 kmax = 250 s-1 at 25 degrees 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 kmax = 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 A resolution using synchrotron radiation. The structure has been solved by MAD phasing.