Biochemical, Mössbauer, and EPR Studies of the Diiron Cluster of Phenol Hydroxylase from Pseudomonas sp. Strain CF 600

Abstract

Phenol hydroxylase of Pseudomonas sp. strain CF600 comprises three components: DmpP is an FAD- and [2Fe-2S]-containing reductase; DmpM is a cofactorless activator protein; and DmpLNO is the oxygenase. Single turnover experiments established that DmpLNO contains the active site, but requires DmpM for efficient turnover: the steady-state turnover rate reaches a maximum at 1.5 DmpM:1 DmpLNO. Chemical cross-linking experiments showed that DmpM interacts with the large subunit of the DmpLNO oxygenase complex. Mössbauer studies revealed that the active site of the oxygenase can accommodate two types of diiron clusters, each of these cluster types having two equivalent sites. Cluster form I, representing typically around 85% of total Fe, has DeltaE(Q) = 1.73 mm/s and delta = 0.54 mm/s, while cluster II exhibits DeltaE(Q) = 0.79 mm/s and delta = 0.48 mm/s. Studies in strong applied magnetic fields suggest that the two iron sites of cluster I are bridged by an oxo group while sites in cluster II appear to be hydroxo-bridged. Reduction of the samples with dithionite yields the diferrous forms of the clusters. Air oxidation of the reduced samples leads to an increase of the cluster II fraction, accompanied by a corresponding decrease in catalytic activity. The reduced oxygenase samples exhibit at X-band an integer spin EPR signal centered, in parallel mode, at g = 16.6. Quantitative analysis showed that 19% of the clusters contribute to the EPR signal, suggesting that cluster II is the EPR-active species. Incubation with dithiothreitol (DTT) inactivated the oxygenase by a mechanism apparently involving H(2)O(2) generation. In addition, Mössbauer studies of DTT-inactivated enzyme showed that all ferric iron belonged to one diamagnetic diferric cluster with parameters that indicate that DTT coordinates to the cluster.

X-ray Structure of a Hydroxylase−Regulatory Protein Complex from a Hydrocarbon-Oxidizing Multicomponent Monooxygenase, Pseudomonas sp. OX1 Phenol Hydroxylase,

“…strain CF600 is 81% identical to the P. stutzeri OX1 enzyme and forms a complex with its regulatory component involving the α-subunit as deduced by chemical cross-linking studies (51). Spectroscopic investigations of this enzyme indicate that the oxidized form contains both (μ-oxo)-and di-μ-(hydroxo)diiron(III) metal centers (12) and that the predominant, catalytically competent species is the oxo-bridged diiron(III) form. Preliminary EPR spectroscopic analysis of samples of P. stutzeri OX1 PHH in its resting and cryo-reduced states reveals that the purified protein contains a heterogeneous mixture of diiron(III) and mixed-valent Fe(II)Fe(III) species, the relative concentrations of which are at present unknown.…”

“…As purified, the enzyme has an optical band at 350 nm that is indicative of a (μ-oxo)diiron(III) species comprising ∼68-96% of the sample (ε 350 = 4800-6000 M −1 cm −1 per dinuclear iron cluster) (12). PHH from Pseudomonas sp.…”

“…Solvent-derived water and hydroxide ions complete the octahedral coordination spheres. Sequence, spectroscopic, and structural analyses suggest that all BMM hydroxylases have nearly identical dinuclear iron centers (1,(10)(11)(12). Because AMO, TMO, SsoMO, and PH are incapable of hydroxylating alkanes, whereas sMMO and THFMO are adept at this transformation, other features of the system must control substrate reactivity at their dioxygen-activated metal centers.…”

Characterization of the Arene-Oxidizing Intermediate in ToMOH as a Diiron(III) Species

“…CF600. 25 The efficiency of the NADH oxidase reaction, defined as the ratio of the decrease in NADH concentration to the increase in hydrogen peroxide concentration, was 51(4)%. Addition of substrate to the reaction mixture retarded peroxide formation, implying that phenol reacts either with a peroxodiiron(III) center or a precursor of such a transient.…”

Construction of Overexpression Vectors and Purification of the Oxygenase Component of Alkylphenol Hydroxylase of Pseudomonas alkylphenolia

“…Modulator protein (about 10 kDa) is required for maximal catalytic activity of substrate hydroxylation (Cadieux and Powlowski, 1999). The oxygenase component at a quaternary structure of (αβγ)2 with a calculated molecular mass of 220 kDa contains a Fe-O-Fe diiron center at the active site and catalyzes hydroxylation reactions in consumption of O2 and electrons provided by reductase (Cadieux et al, 2002). The DmpK has been shown to play a role in assembly of the active form of the oxygenase component of dimethyl PH (Powlowski et al, 1997).…”