Cytochrome c peroxidase activity of a protease-modified form of cytochrome c-552 from the denitrifying bacterium Pseudomonas perfectomarina.
Protease activity present in aerobically grown cells of Pseudomonas perfectomarina, protease apparently copurified with cytochrome c-552, and trypsin achieved a limited proteolysis of the diheme cytochrome c-552. That partial lysis conferred cytochrome c peroxidase activity upon cytochrome c-552. The removal of a 4000-Da peptide explains the structural changes in the cytochrome c-552 molecule that resulted in the appearance of both cytochrome c peroxidase activity (with optimum activity at pH 8.6) and a high-spin heme iron. The oxidized form of the modified cytochrome c-552 bound cyanide to the high-spin ferric heme with a rate constant of (2.1 +/- 0.1) X 10(3) M-1 s-1. The dissociation constant was 11.2 microM. Whereas the intact cytochrome c-552 molecule can be half-reduced by ascorbate, the cytochrome c peroxidase was not reducible by ascorbate, NADH, ferrocyanide, or reduced azurin. Dithionite reduced the intact protein completely but only half-reduced the modified form. The apparent second-order rate constant for dithionite reduction was (7.1 +/- 0.1) X 10(2) M-1 s-1 for the intact protein and (2.2 +/- 0.1) X 10(3) M-1 s-1 for the modified form. In contrast with other diheme cytochrome c peroxidases, reduction of the low-spin heme was not necessary to permit ligand binding by the high-spin heme iron.
Denariaz
C M
CM
Department of Biochemistry, University of Georgia, Athens 30602.
Liu
M Y
MY
Payne
W J
WJ
LeGall
J
J
Marquez
L
L
Dunford
H B
HB
Van Beeumen
J
J
D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D003574:Cytochrome c Group / Q000302:isolation & purification*; D003578:Cytochrome-c Peroxidase / Q000302:isolation & purification*; D004227:Dithionite / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction / Q000187:drug effects; D010446:Peptide Fragments / Q000302:isolation & purification; D010544:Peroxidases / Q000302:isolation & purification*; D011190:Potassium Cyanide / Q000494:pharmacology; D011549:Pseudomonas / Q000201:enzymology*; D013053:Spectrophotometry; D014357:Trypsin
D003574:Cytochrome c Group; D010446:Peptide Fragments; C083951:cytochrome c553; C007955:hemochromogen; D004227:Dithionite; D006418:Heme; C022302:cytochrome C-552; D010544:Peroxidases; D003578:Cytochrome-c Peroxidase; D014357:Trypsin; D011190:Potassium Cyanide; D001205:Ascorbic Acid