Redox Centers of 4-Hydroxybenzoyl-CoA Reductase, a Member of the Xanthine Oxidase Family of Molybdenum-containing Enzymes

Matthias Boll,Georg Fuchs,Christian Meier,Alfred Trautwein,Asma El Kasmi,Stephen W Ragsdale,Grant Buchanan,David J Lowe

doi:10.1074/jbc.m106766200

Abstract

4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a key enzyme in the anaerobic metabolism of phenolic compounds. It catalyzes the reductive removal of the hydroxyl group from the aromatic ring yielding benzoyl-CoA and water. The subunit architecture, amino acid sequence, and the cofactor/metal content indicate that it belongs to the xanthine oxidase (XO) family of molybdenum cofactor-containing enzymes. 4-HBCR is an unusual XO family member as it catalyzes the irreversible reduction of a CoA-thioester substrate. A radical mechanism has been proposed for the enzymatic removal of phenolic hydroxyl groups. In this work we studied the spectroscopic and electrochemical properties of 4-HBCR by EPR and Mössbauer spectroscopy and identified the pterin cofactor as molybdopterin mononucleotide. In addition to two different [2Fe-2S] clusters, one FAD and one molybdenum species per monomer, we also identified a [4Fe-4S] cluster/monomer, which is unique among members of the XO family. The reduced [4Fe-4S] cluster interacted magnetically with the Mo(V) species, suggesting that the centers are in close proximity, (<15 A apart). Additionally, reduction of the [4Fe-4S] cluster resulted in a loss of the EPR signals of the [2Fe-2S] clusters probably because of magnetic interactions between the Fe-S clusters as evidenced in power saturation studies. The Mo(V) EPR signals of 4-HBCR were typical for XO family members. Under steady-state conditions of substrate reduction, in the presence of excess dithionite, the [4Fe-4S] clusters were in the fully oxidized state while the [2Fe-2S] clusters remained reduced. The redox potentials of the redox cofactors were determined to be: [2Fe-2S](+1/+2) I, -205 mV; [2Fe-2S] (+1/+2) II, -255 mV; FAD/FADH( small middle dot)/FADH, -250 mV/-470 mV; [4Fe-4S](+1/+2), -465 mV and Mo(VI)/(V)/(VI), -380 mV/-500 mV. A catalytic cycle is proposed that takes into account the common properties of molybdenum cofactor enzymes and the special one-electron chemistry of dehydroxylation of phenolic compounds.

Highlights

Aromatic compounds comprise a large group of natural products many of which contain hydroxyl or methoxyl functionalities
Together with the measured Fe content of 15–16 iron per protein molecule our results indicate that a dimer of 4-Hydroxybenzoyl-CoA reductase (4-HBCR) contains two [4Fe-4S] clusters and four [2Fe-2S] clusters
In common with all members of this family, monomeric 4-HBCR contains two different [2Fe-2S] centers, a molybdopterin cofactor and a FAD binding subunit that is completely lacking in aldehyde oxidases (10 –12)

Summary

EXPERIMENTAL PROCEDURES

Growth of Bacterial Cells—T. aromatica (DSM 6984) was isolated in our Freiburg laboratory and has been deposited in the Deutsche Sammlung von Mikroorganismen (Braunschweig, Germany) [18]. Excess dithionite and corresponding oxidation products were removed by passing the concentrated enzyme sample (0.1–1 mM; 0.5–1 ml) over a Biogel P-6 (Bio-Rad) desalting column (volume: 5 ml, diameter: 0.7 cm) equilibrated with either 100 mM Mops/KOH, pH 7.5, or 100 mM Hepes/HCl, pH 8.3, both containing 10 mM MgCl2. Reduction and Oxidation of the Enzyme—Reduction of 4-HBCR was performed by adding sodium dithionite from a freshly prepared stock solution (100 mM in 100 mM Mops/KOH, pH 7.5) giving a final 10-fold excess of this reductant compared with the enzyme. Protein was visualized by Coomassie Blue staining [25]

RESULTS

EPR conditions

DISCUSSION