Crystallization of 4-hydroxybenzoyl-CoA reductase and the structure of its electron donor ferredoxin.
4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a central enzyme in the metabolism of phenolic compounds in anaerobic bacteria. The enzyme catalyzes the reductive removal of the phenolic hydroxyl group from 4-hydroxybenzoyl-CoA, yielding benzoyl-CoA and water. 4-HBCR belongs to the xanthine oxidase (XO) family of molybdenum enzymes which occur as heterodimers, (alphabetagamma)(2). 4-HBCR contains two molybdopterins, four [2Fe-2S] and two [4Fe-4S] clusters and two FADs. A low-potential Allochromatium vinosum-type ferredoxin containing two [4Fe-4S] clusters serves as an in vivo electron donor for 4-HBCR. In this work, the oxygen-sensitive proteins 4-HBCR and the ferredoxin (TaFd) from Thauera aromatica were crystallized under anaerobic conditions. 4-HBCR crystallized with PEG 4000 and MPD as precipitant diffracted to about 1.6 A resolution and the crystals were highly suitable for X-ray structure analysis. Crystals of TaFd were obtained with (NH(4))(3)PO(4) as precipitant and revealed a solvent content of 77%, which is remarkably high for a small soluble protein. The structure of TaFd was solved at 2.9 A resolution by the molecular-replacement method using the highly related structure of the ferredoxin (CvFd) from A. vinosum as a model. Structural changes between the two ferredoxins around the [4Fe-4S] cluster can be correlated with their different redox potentials.
Unciuleac
Mihaela
M
Institut für Biologie II, Mikrobiologie, Schänzlestrasse 1, D-79104 Freiburg, Germany.
Boll
Matthias
M
Warkentin
Eberhard
E
Ermler
Ulrich
U
D018360:Crystallography, X-Ray / Q000379:methods*; D005288:Ferredoxins / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D020628:Thauera / Q000201:enzymology*; D014969:Xanthine Oxidase / Q000737:chemistry
D005288:Ferredoxins; D014969:Xanthine Oxidase; D044925:Oxidoreductases Acting on CH-CH Group Donors; C060482:benzoyl-coenzyme A-4-oxidoreductase