An electron-spin-resonance study on the redox-active centers of the 4-methoxybenzoate monooxygenase from Pseudomonas putida.

Abstract

4-Methoxybenzoate monooxygenase isolated from a species of pseudomonas putida can be separated in two components, NADH-putidamonooxin oxidoreductase and putidamonooxin, the latter contains the dioxygen activating site. Binding of tight coupling substrates leads to monooxygenase activity of this enzyme system with substrate hydroxylation, whereas binding of uncoupling substrates leads to oxidase activity with production of H2O2. The NADH-putidamonooxin oxidoreductase when reduced with NADH exhibits an electron spin resonance (ESR) spectrum with rhombic symmetry and an isotropic resonance at g = 2.0033. The average g value of 1.96 and the temperature behaviour of the rhombic spectrum are indicative for (2Fe-2S) clusters. In the range of pH 6.8-8.2 the isotropic ESR signal attributed to FMN shows the characteristic linewidth of the blue semiquinone form. The reduced putidamonooxin shows an ESR spectrum with rhombic symmetry and an average g value of 1.89 caused by a pronounced g anisotropy. An identical g anisotropy is observed for the Rieske-cluster. The 57Fe-enriched (2Fe-2S) clusters show hyperfine interaction between the electron spin and the nuclear spin of the iron atoms in two principal directions of the iron atoms in two principal directions of the A-tensor, the resonance in the third direction is not resolved. For one direction equivalent hyperfine constants with a = 2.0 mT and a<0.05 mT respectively indicating different spin densities at the iron atoms in the cluster. Hyperfine constants and g tensor of the reduced cluster are not influenced by binding of putidamonooxin. The ESR feature in the range of g = 9.6-4.3 is characteristic for a mononuclear non-heme iron center in oxidized putidamonooxin. The temperature behaviour of the resonance at g5= 4.293 in the range of 3.6-80 K indicates a zero-field splitting with D=0.90.± 0.1cm−1.Therefore, we assume that this iron center is probably coordinated by oxygen and nitrogen atoms. On the basis of g value this mononuclear non-herme iron center has at least two different microenvironments generating rhombic (E/D= 0.33) and tetragonal symmetry (E/D= 0.05-0.15). Whereas the iron with tetragonal symmetry is sensitive to substrate-binding. The iron with rhombic symmetry seems to b eindependent of substrate-binding. Nevertheless, both symmetry species are involved in the aaactivation of dioxygen as the iron-sulfur cluster can only be aerobically oxidized if mono nuclear iron centers are present in a 1:1 ratio. Substitution of the mononuclear non-hemr iron center by 57Fe leads to a line-broadening of the resonance at g5= 4.293 indicating a splitting constant of 0.8 + 0.2 mT leads to a line-broadening of the resonance at g5=4.293 indicating a splitting constant of 0.8±0.2mT. It is excluded, by comparing aerobically and anaerobically oxidized putidamonooxin, that the iron center with tetragonal symmetry belongs to a ternary dioxygen enzyme substrate complex.