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

Abstract

The gene encoding the alkane -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 units͞mg 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) ⌬ 9 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 O 2 and substrate (octane) were added, however, the diferrous cluster was quantitatively reoxidized, proving that the diiron cluster occupies the active site. Mössbauer 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 O 2 -activation biochemistry.

Domain Swapping Localizes the Structural Determinants of Regioselectivity in Membrane-bound Fatty Acid Desaturases of Caenorhabditis elegans

“…Sequence comparisons, site-directed mutagenesis, and spectroscopic results are consistent with histidine-rich coordination of a diiron center at the active site of these enzymes (10,30). Eight highly conserved histidine residues have been shown to be required for function with the exception of one that is replaced by glutamine in some "front-end" desaturases, which introduce double bonds near C-1 in the substrate (3,31).…”

Organophosphonate-degrading PhnZ reveals an emerging family of HD domain mixed-valent diiron oxygenases

“…The signals are broad and suggest the presence of two or more Fe II complexes (SI Appendix, Fig. S2), as has been seen in MIOX and other nonheme diiron proteins (16,(27)(28)(29).…”

A family of diiron monooxygenases catalyzing amino acid beta-hydroxylation in antibiotic biosynthesis

“…Accordingly, the Mössbauer spectrum of reduced CmlA more closely resembles those of Δ9D, MMO, and RNR (1 N and 3-5 O ligands per iron) with a similar isomer shift δ ≥ 1.24 (21,25,37). Diferrous enzymes with more nitrogen ligands such as hemerythrin (2 N and 3 O ligands per iron, δ ¼ 1.14 mm∕s) and AlkB (4 N ligands per iron, δ ¼ 1.05-1.15 mm∕s) produce substantially smaller isomer shift values (38,39). Based on the conserved sequence motif of CmlA homologs and these spectroscopic parameters, a model for the dinuclear iron site is presented in Fig.…”