Mössbauer studies of alkane omega-hydroxylase: evidence for a diiron cluster in an integral-membrane enzyme.
The gene encoding the alkane omega-hydroxylase (AlkB; EC 1.14.15.3) from Pseudomonas oleovorans was expressed in Escherichia coli. The integral-membrane protein was purified as nearly homogeneous protein vesicles by differential ultracentrifugation and HPLC cation exchange chromatography without the detergent solubilization normally required for membrane proteins. Purified AlkB had specific activity of up to 5 units/mg for octane-dependent NADPH consumption. Mössbauer studies of AlkB showed that it contains an exchange-coupled dinuclear iron cluster of the type found in soluble diiron proteins such as hemerythrin, ribonucleotide reductase, methane monooxygenase, stearoyl-acyl carrier protein (ACP) delta9 desaturase, rubrerythrin, and purple acid phosphatase. In the as-isolated enzyme, the cluster contains an antiferromagnetically coupled pair of high-spin Fe(III) sites, with an occupancy of up to 0.9 cluster per AlkB. The diferric cluster could be reduced by sodium dithionite, and the diferrous state was found to be stable in air. When both O2 and substrate (octane) were added, however, the diferrous cluster was quantitatively reoxidized, proving that the diiron cluster occupies the active site. Mossbauer data on reduced AlkB are consistent with a cluster coordination rich in nitrogen-containing ligands. New sequence analyses indicate that at least 11 nonheme integral-membrane enzymes, including AlkB, contain the 8-histidine motif required for catalytic activity in stearoyl-CoA desaturase. Based on our Mössbauer studies of AlkB, we propose that the integral-membrane enzymes in this family contain diiron clusters. Because these enzymes catalyze a diverse range of oxygenation reactions, this proposal suggests a greatly expanded role for diiron clusters in O2-activation biochemistry.
Shanklin
J
J
Department of Biology, Brookhaven National Laboratory, Upton, NY 11973, USA.
Achim
C
C
Schmidt
H
H
Fox
B G
BG
Münck
E
E
D007501:Iron / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics; D011549:Pseudomonas / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D015204:Spectroscopy, Mossbauer
D008565:Membrane Proteins; D011994:Recombinant Proteins; D007501:Iron; D006899:Mixed Function Oxygenases