Studies of electron-transfer properties of salicylate hydroxylase from Pseudomonas cepacia and effects of salicylate and benzoate binding.
The pH dependence of the redox behavior of salicylate hydroxylase from Pseudomonas cepacia as well as the effects of salicylate, benzoate, and chloride binding is described. At pH 7.6 in 0.02 M potassium phosphate buffer E1(0')(EFl ox/EFl.-) is -0.150 V and E2(0')(EFl.-/EFl red H-) is -0.040 V versus the standard hydrogen electrode (SHE). A maximum of 5% of FAD anion semiquinone is thermodynamically stabilized under these conditions. However, in coulometric and dithionite titrations more semiquinone is kinetically formed, indicating slow transfer of the second electron. The potential/pH dependence is consistent with a two-electron, one-proton transfer. Upon salicylate binding the midpoint potential is shifted 0.020 V negative from -0.094 to -0.114 V vs SHE at pH 7.6. A maximum of 7% of the neutral semiquinone is stabilized both in potentiometric and coulometric titrations. This small potential shift indicates that the substrate is bound nearly to the same extent to all three oxidation states of the enzyme. It is clear that the substrate binding does not make the reduction of the flavin thermodynamically more favorable. In contrast to salicylate, the potential shift caused by the effector, benzoate, is much more significant. (A maximum potential shift of -0.07 V is calculated.) Benzoate binds most tightly to the oxidized form and is least tightly bound to the two-electron-reduced form of the enzyme. For the reduction of the free enzyme the transfer of the second electron or the transfer of the proton is rate limiting, as is shown by the kinetic formation of the anionic semiquinone.(ABSTRACT TRUNCATED AT 250 WORDS)
Einarsdottir
G H
GH
Department of Chemistry, University of Minnesota, Minneapolis 55455.
Stankovich
M T
MT
Tu
S C
SC
D001565:Benzoates / Q000494:pharmacology*; D019817:Benzoic Acid; D002712:Chlorides / Q000494:pharmacology; D004227:Dithionite / Q000494:pharmacology; D004579:Electron Transport; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010084:Oxidation-Reduction; D011199:Potentiometry; D011485:Protein Binding; D011549:Pseudomonas / Q000201:enzymology*; D012459:Salicylates / Q000494:pharmacology*
D001565:Benzoates; D002712:Chlorides; D012459:Salicylates; D004227:Dithionite; D019817:Benzoic Acid; D006899:Mixed Function Oxygenases; C022619:salicylate 1-monooxygenase