Redox centers of 4-hydroxybenzoyl-CoA reductase, a member of the xanthine oxidase family of molybdenum-containing enzymes.
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.
Boll
M
M
Institut für Biologie II, Mikrobiologie, Schänzlestrasse 1, D-79104 Freiburg, Germany. boll@uni-freiburg.de
Fuchs
G
G
Meier
C
C
Trautwein
A
A
El Kasmi
A
A
Ragsdale
S W
SW
Buchanan
G
G
Lowe
D J
DJ
D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D008982:Molybdenum / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D015204:Spectroscopy, Mossbauer; D014969:Xanthine Oxidase / Q000378:metabolism*
D007506:Iron-Sulfur Proteins; D005182:Flavin-Adenine Dinucleotide; C029276:flavin semiquinone; D008982:Molybdenum; D010088:Oxidoreductases; D014969:Xanthine Oxidase; D044925:Oxidoreductases Acting on CH-CH Group Donors; C060482:benzoyl-coenzyme A-4-oxidoreductase