MOSSBAUER STUDIES OF ALKANE OMEGA-HYDROXYLASE - EVIDENCE FOR A DIIRONCLUSTER IN AN INTEGRAL-MEMBRANE ENZYME

The gene encoding the alkane omega-hydroxylase (AlkB; EC 1.14.15.3) fr om Pseudomonas oleovorans was expressed in Escherichia coli, The integ ral-membrane protein was purified as nearly homogeneous protein vesicl es by differential ultracentrifugation and HPLC cation exchange chroma tography without the detergent solubilization normally required for me mbrane proteins, Purified AlkB had specific activity of up to 5 units/ mg for octane-dependent NADPH consumption, Mossbauer studies of AlkB s howed that it contains an exchange-coupled dinuclear iron cluster of t he type found in soluble diiron proteins such as hemerythrin, ribonucl eotide reductase, methane monooxygenase, stearoyl-acyl carrier protein (ACP) Delta(9) desaturase, rubrerythrin, and purple acid phosphatase, In the as-isolated enzyme, the cluster contains an antiferromagnetica lly coupled pair of high-spin Fe(lII) sites, with an occupancy of up t o 0.9 cluster per AlkB, The diferric cluster could be reduced by sodiu m dithionite, and the diferrous state was found to be stable in air, W hen both O-2 and substrate (octane) were added, however, the diferrous cluster was quantitatively reoxidized, proving that the diiron cluste r occupies the active site, Mossbauer data on reduced AlkB are consist ent with a cluster coordination rich in nitrogen-containing ligands, N ew sequence analyses indicate that at least 11 nonheme integral-membra ne enzymes, including AlkB, contain the 8-histidine motif required for catalytic activity in stearoyl-CoA desaturase, Based on our Mossbauer studies of AlkB, we propose that the integral-membrane enzymes in thi s family contain diiron clusters, Because these enzymes catalyze a div erse range of oxygenation reactions, this proposal suggests a greatly expanded role for diiron clusters in O-2-activation biochemistry.