Sulfolobus acidocaldarius terminal oxidase. A kinetic investigation and its structural interpretation.
The thermoacidophilic archaebacterium Sulfolobus acidocaldarius possesses a very unusual terminal oxidase. We report original kinetic experiments on membranes of this microorganism carried out by stopped flow, using time-resolved optical spectroscopy combined with singular value decomposition analysis. The reduced-oxidized kinetic difference spectrum of the Sulfolobus membranes is characterized by three significant peaks in the visible region at 605, 586, and 560 nm. The 605-nm peak and part of the 586-nm peak (cytochrome aa3-type quinol oxidase) are reduced synchronously by both ascorbate plus N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) and dithionite, and they are very rapidly oxidized by molecular oxygen. A second pool of cytochromes seems to contribute to the 586-nm peak which is not reduced by ascorbate plus TMPD and reacts very slowly with dithionite. The b-type cytochromes (560 nm peak) are reduced by both reductants and are essentially "non-autoxidizable" at room temperature. Only one CO binding site with spectral features, kinetic properties, and ligand affinity not very dissimilar from those of mammalian cytochrome oxidase can be detected in the ascorbate-reduced membranes. On the contrary, a second CO binding site having unusual properties for aa3 terminal oxidases can be detected in the dithionite-reduced membranes.
Giuffrè
A
A
Department of Biochemical Sciences, University of Rome, La Sapienza, Italy.
Antonini
G
G
Brunori
M
M
D'Itri
E
E
Malatesta
F
F
Nicoletti
F
F
Anemüller
S
S
Gleissner
M
M
Schäfer
G
G
D001205:Ascorbic Acid / Q000737:chemistry; D002248:Carbon Monoxide / Q000378:metabolism; D004227:Dithionite / Q000737:chemistry; D007700:Kinetics; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D016945:Sulfolobus acidocaldarius / Q000201:enzymology*; D013772:Tetramethylphenylenediamine / Q000737:chemistry
D004227:Dithionite; D002248:Carbon Monoxide; D010088:Oxidoreductases; D013772:Tetramethylphenylenediamine; D001205:Ascorbic Acid; D010100:Oxygen