Cytochrome-c(552) from Thermus thermophilus: A functional and crystallographic investigation

The eubacterium Thermus thermophilus expresses terminal oxidases of the ba(3)- and caa(3)-type. The soluble cytochrome-c(552) 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 ba(3)-oxidase. In the presence of TMPD and ascorbate, biphasic Eadie-Hofstee plots with k(max) = 250 s(-1) at 25 degrees C are observed upon addition of cytochrome-c(552). Surprisingly, the caa(3)-oxidase with its single covalently bound cytochrome-e also exhibits a biphasic redox activity with k(max) = 185 s(-1) in the presence of TMPD and ascorbate only. Further addition of cytochrome-c(552) does not lead to enhanced activity. Crystals of cytochrome-c(552) were obtained by vapor diffusion using the sitting-drop method in the presence of ammonium sulfate as precipitant. They diffract to 1.28 Angstrom resolution using synchrotron radiation. The structure has been solved by MAD phasing. (C) 1997 Academic Press.