title: Iron content of human 5-lipoxygenase, effects of mutations regarding conserved histidine residues.
abstract: Recombinant human 5-lipoxygenase was expressed in Escherichia coli and purified to more than 95% homogeneity by ammonium sulfate precipitation and agarose-ATP column chromatography. The specific activity of the purified enzyme was 21-28 mumol/mg, as assessed by the generation of 5-hydro(pero)xyeicosatetraenoic acid. The iron content was analyzed by graphite furnace atomic absorption spectrophotometry for six preparations of the enzyme. The average value of the iron content was 0.86 mol/mol (iron/protein) with a range of 0.74-1.15 mol/mol. All lipoxygenases that have been sequenced contain 6 conserved histidine residues. Mutants of 5-lipoxygenase, with substitutions of these 6 conserved histidines, were purified and analyzed. Mutants H372Q and H550Q had no detectable enzyme activity and were also practically devoid of iron. Three mutants regarding His367 (H367Q, H367N, and H367S) were all inactive but had partial iron contents (0.5, 0.2, and 0.5 mol/mol, respectively). Finally, the mutated proteins H362Q, H390Q, and H399Q displayed reduced enzyme activity but contained similar amounts of iron as non-mutated 5-lipoxygenase. We conclude that histidines 372 and 550 constitute two of the iron ligands in 5-lipoxygenase. Also His367 is necessary for the enzyme activity, but this residue is not crucial for binding of iron.
authors.affiliation: Department of Physiological Chemistry, Karolinska Institute, Stockholm, Sweden.
authors.forename: Y Y
authors.initials: YY
authors.lastname: Zhang
authors.forename: B
authors.initials: B
authors.lastname: Lind
authors.forename: O
authors.initials: O
authors.lastname: Rådmark
authors.forename: B
authors.initials: B
authors.lastname: Samuelsson
mesh_terms: D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002845:Chromatography / Q000379:methods; D006639:Histidine / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship
chemical_list: D008024:Ligands; D011994:Recombinant Proteins; D006639:Histidine; D001094:Arachidonate 5-Lipoxygenase