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.
Soulimane
T
T
Institut für Biochemie, Klinikum der RWTH Aachen, Germany.
von Walter
M
M
Hof
P
P
Than
M E
ME
Huber
R
R
Buse
G
G
D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000494:pharmacology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D018360:Crystallography, X-Ray; D003573:Cytochrome b Group / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D005285:Fermentation; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000737:chemistry / Q000254:growth & development; D014462:Ultrafiltration
D003573:Cytochrome b Group; D003574:Cytochrome c Group; C083951:cytochrome c553; C022302:cytochrome C-552; C058557:cytochrome ba3; D003576:Electron Transport Complex IV; D001205:Ascorbic Acid