Mössbauer studies of alkane ω-hydroxylase: Evidence for a diiron cluster in an integral-membrane enzyme

• DOI: 10.1073/pnas.94.7.2981
• Publication Date: 2002-07-26T14:35:50Z
• Authors: John Shanklin; Catalina Achim; Hermann Schmidt; Brian G. Fox; Eckard Münck
• Keywords: AlkB; Methane Monooxygenase

The gene encoding the alkane ω-hydroxylase (AlkB; EC 1.14.15.3 ) from Pseudomonas oleovorans was expressed in Escherichia coli . integral-membrane protein purified as nearly homogeneous vesicles by differential ultracentrifugation and HPLC cation exchange chromatography without detergent solubilization normally required for membrane proteins. Purified AlkB had specific activity of up to 5 units/mg octane-dependent NADPH consumption. Mössbauer studies showed that it contains an exchange-coupled dinuclear iron cluster type found soluble diiron proteins such hemerythrin, ribonucleotide reductase, methane monooxygenase, stearoyl-acyl carrier (ACP) Δ 9 desaturase, rubrerythrin, purple acid phosphatase. In as-isolated enzyme, antiferromagnetically coupled pair high-spin Fe(III) sites, with occupancy 0.9 per AlkB. diferric could be reduced sodium dithionite, diferrous state stable air. When both O 2 substrate (octane) were added, however, quantitatively reoxidized, proving occupies active site. data on are consistent a coordination rich nitrogen-containing ligands. New sequence analyses indicate at least 11 nonheme enzymes, including AlkB, contain 8-histidine motif catalytic stearoyl-CoA desaturase. Based our we propose enzymes this family clusters. Because these catalyze diverse range oxygenation reactions, proposal suggests greatly expanded role clusters -activation biochemistry.