Open AccessKinetics of Reduction of Putidamonooxin by NADH–Putidamonooxin Oxidoreductase, Sodium Dithionite and Superoxide Radicals
Author(s) -
BERNHARDT FrithjofHans,
KUTHAN Hartmut
Publication year - 1983
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1983.tb07122.x
Subject(s) - oxidoreductase , chemistry , dithionite , sodium dithionite , heme , reductase , cofactor , substrate (aquarium) , photochemistry , biochemistry , inorganic chemistry , enzyme , biology , ecology
Putidamonooxin, a conjugated iron‐sulfur protein, functions as the oxygen‐activating component within the 4‐methoxybenzoate monooxygenase from Pseudomonas putida . A[2Fe–2S] cluster and a mononuclear non‐heme iron together form the oxygen‐activating units within the active sites of putidamonooxin. In the present paper the reduction kinetics of oxidized putidamonooxin were studied in the absence and in the presence of a substrate and/or the mononuclear non‐heme iron. The reducing agenst used were (a) NADH plus the NADH–putidamonooxin oxidoreductase and (b) artificial reductants, i.e. O 2 radicals and sodium dithionite in the presence or in the absence of the NADH‐putidamonooxin oxidoreductase. The reduction kinetics of cofactor‐iron‐depleted putidamonooxin revealed that the addition of a substrate strongly enhanced the rate of reduction of the [2Fe–2S] chromophores by the native reductase but significantly decreased the reduction by artificial reductants. These effects were even more pronounced when the mononuclear non‐heme iron was bound to putidamonooxin in addition to the substrate. The presence of the native reductase markedly enhanced the rate of reduction of native putidamonooxin by sodium dithionite, though not to the optimal rate as measured in the presence of both NADH and the NADH–putidamonooxin oxidoreductase. The experimental findings are taken to show that the reduction of oxidized putidamonooxin in the absence and in the presence of its mononuclear non‐heme iron, when NADH–putidamonooxin oxidoreductase and artificial reductants are used as electron donors, may occur at two different sites, both of which are influenced by substrate binding. The most powerful reductant of putidamonooxin is the NADH–putidamonooxin oxidoreductase in its partially reduced state (which is characterized by its broad absorption band centered near 600 nm and its typical isotropic electron‐paramagnetic‐resonance signal with g = 2.0033), rather than in its fully reduced state.