Multiple roles of component proteins in bacterial multicomponent monooxygenases: phenol hydroxylase and toluene/o-xylene monooxygenase from Pseudomonas sp. OX1

Abstract

Phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO) from Pseudomonas sp. OX1 require three or four protein components to activate dioxygen for the oxidation of aromatic substrates at a carboxylate-bridged diiron center. In this study, we investigated the influence of the hydroxylases, regulatory proteins, and electron-transfer components of these systems on substrate (phenol; NADH) consumption and product (catechol; H(2)O(2)) generation. Single-turnover experiments revealed that only complete systems containing all three or four protein components are capable of oxidizing phenol, a major substrate for both enzymes. Under ideal conditions, the hydroxylated product yield was ∼50% of the diiron centers for both systems, suggesting that these enzymes operate by half-sites reactivity mechanisms. Single-turnover studies indicated that the PH and ToMO electron-transfer components exert regulatory effects on substrate oxidation processes taking place at the hydroxylase actives sites, most likely through allostery. Steady state NADH consumption assays showed that the regulatory proteins facilitate the electron-transfer step in the hydrocarbon oxidation cycle in the absence of phenol. Under these conditions, electron consumption is coupled to H(2)O(2) formation in a hydroxylase-dependent manner. Mechanistic implications of these results are discussed.

Figure 1

Representative profiles of H₂O₂ generated by PH (a) and ToMO (b). (a) H₂O₂ generation profile upon addition of 200 μM NADH to a solution of 1 μM PHH, 6 μM PHM, 2 μM PHP, and 10 μM NH₂OH at pH 7.5 and 4 °C in the presence (diamonds, solid lines) or absence (circles, dashed lines) of 5 mM phenol. (b) H₂O₂ generation profile upon addition of 200 μM NADH to a solution of 1 μM ToMOH, 2 μM ToMOD, 2 μM ToMOC, 0.1 μM ToMOF, and 10 μM NH₂OH at pH 7.5 and 4 °C in the presence (diamonds, solid lines) or absence (circles, dashed lines) of 5 mM phenol. Data obtained in the absence of phenol were fit (solid lines) to the single exponential formation process y = A*exp(−kt) + B. Reaction solutions were assayed for H₂O₂ content as noted in the text.

Figure 2

Percent PH activity remaining as a function of time following incubation with NADH. Reaction solutions containing 1 μM PHH, 6 μM PHM, 2 μM PHP in 500 μL of 0.1 M Tris-HCl, pH 7.5, were incubated with 5 mM NADH for a specified time period between 0 and 8 min, after which 5 mM phenol was added. Reactions were allowed to proceed for 20 min and then were quenched by addition of 100 μL of TCA. Catechol content was monitored by HPLC. Data are plotted as the percentage of the amount of catechol formed in experiments in which NADH and phenol were added simultaneously (t = 0 min) versus the time that the reaction mixture was incubated with NADH before addition of phenol. Data points represent the average of two trials performed with different batches of protein.

Figure 3

(a) Representative profile of H₂O₂ generation (diamonds) upon addition of 200 μM NADH to 2 μM PHP at pH 7.5 and 4 °C. Similar reaction profiles were obtained in the presence of NH₂OH. (b) Representative profile of H₂O₂ generation (diamonds) upon addition of 200 μM NADH to a solution of 1 μM PHH, 6 μM PHM, 0.1 μM PHP, and 10 μM NH₂OH at pH 7.5 and 4 °C. Data were fit (solid lines) to the single exponential formation process y = A*exp(−kt) + B. Reaction solutions were assayed for H₂O₂ content as noted in the text. See text for comment on the non-zero ordinate intercepts.

Figure 4

Representative H₂O₂ generation profile upon addition of 200 μM NADH to a solution of 1 μM ToMOH I100W, 2 μM ToMOD, 2 μM ToMOC, 0.1 μM ToMOF, and 10 μM NH₂OH at pH 7.5 and 25 °C in the absence of phenol. Data were fit (solid lines) to a single exponential formation process, y = A*exp(−kt) + B. Reaction solutions were assayed for H₂O₂ content as noted in the text.

Scheme 1

Pathway of benzene metabolism by Pseudomonas sp. OX1.

Scheme 2

Product evolution by PH or ToMO in the presence (i) or absence (ii) of phenol and by PHP in the absence of phenol (iii).

Scheme 3

Proposed mechanism of O₂ activation by ToMO and PH.