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. K_s 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 K_s values here obtained and the previously reported K_m values are discussed.