Chemical and Spectral Properties of Putidamonooxin, the Iron‐Containing and Acid‐Labile‐Sulfur‐Containing Monooxygenase of a 4‐Methoxybenzoate O‐Demethylase from Pseudomonas putida

Abstract

Gel chromatography indicates that putidamonooxin has a molecular weight of about 126000. On the other hand, the amino acid composition and the iron‐to‐protein ratio point to a minimal molecular weight of 33000 and 31000 respectively. On sodium dodecylsulfate/polyacrylamide gel electrophoresis the enzyme migrated as a homogeneous band corresponding to a molecular weight of about 40000. The number of spots found in the tryptic peptide map of the carboxymethylated and digested enzyme indicates that putidamonooxin is composed of three or four identical subunits. After covalent cross‐linking of the subunits with dimethyl suberimidate and subsequent dodecylsulfate electrophoresis the main bands were in the molecular weight range of 40000, 87000 and 124000. These findings lead us to propose that putidamonooxin is either a trimer or tetramer. The amino acid composition of putidamonooxin and related data calculated from this are given. The isoelectric point was shown by isoelectric focusing to be at pH 4.7. Low‐temperature optical spectra of the reduced and oxidized enzyme as well as of three different putidamonooxin substrate complexes are given together with those recorded at 10°C. Enzyme · substrate binding spectra are observed with the oxidized putidamonooxin but not with the reduced enzyme. For the oxidized putidamonooxin a molar absorption coefficient at 455 nm of 14.7 mM−1 cm−1 was determined. Ks values of putidamonooxin towards different substrates and substrate analogues (i.e. tight couplers, partial uncouplers and uncouplers) are presented and possible reasons for the difference between the Ks values here obtained and the previously reported Km values are discussed.

An Electron-Spin-Resonance Study on the Reodex-Active Centers of the 4-Methoxybenzoate Monooxygenase from Pseudomonas putida

“…3 with a reduced amplitude. Binding of substrate to iron-depleted putidainonooxin as proved by visible absosption spectroscopy [3] does not influence this ESR spectrum. The incorporation of additional mononuclear non-heme iron centers strongly bound to its binding-site only in the presence of substrate leads to an increase in intensity of the resonance at g = 4.3.…”

“…Partial uncouplers allow both types of reactions [4,6]. Chemical [2,3] and spectroscopic [7 -101 analysis revealed that both enzyme components contain iron-sulfur clusters as redox-active centers.…”

“…tase with a molecular mass of about 42000 Da and of an oxygenase which is the oligomeric putidamonooxin with a molecular mass of 126000 Da [2,3]. The enzyme system is not highly specific, because different derivatives of benzoic acid can be bound, indicated either by distinct changes in the visible absorption spectra of the oxidized putidamonooxin or by following the dioxygen consumption and NADH oxidation [I, 3 -61.…”

Assay, Purification, and Partial Characterization of Choline Monooxygenase from Spinach

“…Moreover, the extinction coefficients for these enzymes at their respective A maxima are comparable. The value for the P. putidu monooxygenase at 455 nm is 14.7 mM-l cm-' (Bernhardt et al, 1978); that for CMO at 459 nm was estimated as 7 to 11 mM-' cm-' in different preparations, assuming the native molecular mass given below and using A,,, to estimate protein. These CMO values would be underestimates if the chromophore were damaged during protein purification.…”

“…The loss of color upon addition of dithionite is also consistent with an Fe-S protein (Lovenberg, 1973). Although Fe-S proteins typically have a peak near 415 nm and CMO does not, the known exceptions include a monooxygenase from Pseudomonas putida (Bernhardt et al, 1978). The oxidized spec- Wavelength (nm) Figure 6.…”