The alpha subunit of toluene dioxygenase from Pseudomonas putida F1 can accept electrons from reduced FerredoxinTOL but is catalytically inactive in the absence of the beta subunit

Abstract

The oxygenase component of toluene dioxygenase from Pseudomonas putida F1 is an iron-sulfur protein (ISPTOL) consisting of alpha (TodC1) and beta (TodC2) subunits. Purified TodC1 gave absorbance and electron paramagnetic resonance spectra identical to those given by purified ISPTOL. TodC1 was reduced by NADH and catalytic amounts of ReductaseTOL and FerredoxinTOL. Reduced TodC1 did not oxidize toluene, and catalysis was strictly dependent on the presence of purified TodC2.

FIG. 1

Electron flow in the TDO system.

FIG. 2

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of samples taken during the purification of TodC1. Fractions obtained from different steps were analyzed on 12% polyacrylamide gels stained with Coomassie blue. Lanes: M, molecular mass standards; 1, cell extract (10 μg of protein); 2, Q-Sepharose column fraction containing TodC1 (7 μg of protein); 3, hydroxyapatite column fraction containing TodC1 (5 μg of protein).

FIG. 3

Anaerobic reduction of TodC1. Absorption spectra were recorded on an Aminco DW2000 UV-visible spectrophotometer. Cuvettes were rendered anaerobic by alternately flushing and evacuating them with argon. Reaction mixtures contained TodC1 (30 nmol) in 1 ml of MES buffer (pH 6.8). Curve 1 shows the oxidized spectrum of TodC1. The curves between 1 and 2 are spectra of TodC1 obtained by anaerobic additions of NADH (2.5 nmol of each) in the presence of catalytic amounts of TodB (ferredoxin_TOL, 90 μg of protein) and TodA (reductase_TOL, 45 μg of protein). The inset shows anaerobic reduction of TodC1 by NADH in the presence of TodB and TodA. The decrease in absorbance for TodC1 was monitored at 446 nm.