EPR spectroscopy and electrospray ionization mass spectrometry reveal distinctive features of the iron site in leukocyte 12-lipoxygenase.
The procedure for the expression and purification of recombinant porcine leukocyte 12-lipoxygenase using Escherichia coli [K.M. Richards, L.J. Marnett, Biochemistry 36 (1997) 6692-6699] was updated to make it possible to produce enough protein for physical measurements. Electrospray ionization tandem mass spectrometry confirmed the amino acid sequence. The redox properties of the cofactor iron site were examined by EPR spectroscopy at 25K following treatment with a variety of fatty acid hydroperoxides. Combination of the enzyme in a stoichiometric ratio with the hydroperoxides led to a g4.3 signal in EPR spectra instead of the g6 signal characteristic of similarly treated soybean lipoxygenase-1. Native 12-lipoxygenase was also subjected to electrospray ionization mass spectrometry. There was evidence for loss of the mass of an iron atom from the protein as the pH was lowered from 5 to 4. Native ions in these samples indicated that iron was lost without the protein completely unfolding.
Rapp
Johanna
J
Department of Chemistry, University of Toledo, 2801 West Bancroft Street, Toledo, OH 43606, USA.
Xu
Shu
S
Sharp
Allan M
AM
Griffith
Wendell P
WP
Kim
Yong-Wah
YW
Funk
Max O
MO
D000595:Amino Acid Sequence; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism*; D007962:Leukocytes / Q000378:metabolism*; D008054:Lipid Peroxides / Q000494:pharmacology; D008970:Molecular Weight; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D034421:Sus scrofa; D013696:Temperature; D014170:Transformation, Genetic
D008054:Lipid Peroxides; D011994:Recombinant Proteins; D007501:Iron; D001092:Arachidonate 12-Lipoxygenase
J Bacteriol. 1987 Feb;169(2):751-7
3027045
Chem Biol. 2007 May;14(5):473-88
17524979
J Biol Chem. 1958 May;232(1):193-202
13549410
Biochemistry. 1994 Apr 5;33(13):3974-9
8142401
J Biol Chem. 1991 Jan 25;266(3):1597-604
1671037
Eur J Biochem. 1990 Jul 5;190(3):611-7
2164931
J Am Chem Soc. 2004 Feb 25;126(7):2006-15
14971933
Biochem Biophys Res Commun. 2001 Jun 15;284(3):563-7
11396936
Biochemistry. 2001 May 29;40(21):6371-8
11371199
Proteins. 1997 Sep;29(1):15-31
9294864
N Engl J Med. 2007 Nov 1;357(18):1841-54
17978293
Biochemistry. 1997 Jun 3;36(22):6692-9
9184149
Science. 2001 Nov 30;294(5548):1871-5
11729303
Lipids. 1993 Mar;28(3):241-4
8385258
J Am Chem Soc. 2008 Apr 16;130(15):5302-10
18348525
Biochemistry. 2002 Aug 13;41(32):10297-303
12162745
Biomacromolecules. 2002 Jul-Aug;3(4):649-54
12099806
FEBS Lett. 1975 Aug 1;56(1):50-4
169156
Eur J Biochem. 1979 Jun 1;96(3):545-61
111925
Proteins. 2008 Feb 15;70(3):650-8
17879349
Annu Rev Pharmacol Toxicol. 2009;49:265-90
18851701
Biochim Biophys Acta. 1992 Oct 30;1128(2-3):117-31
1420284
Biochim Biophys Acta. 1981 Jan 30;667(1):77-86
6260216
Biochemistry. 2004 Jun 8;43(22):7151-61
15170352
FEBS Lett. 1991 Aug 5;287(1-2):105-7
1652456
Biochem J. 2002 Aug 1;365(Pt 3):865-71
11985498
Nat Struct Biol. 1997 Dec;4(12):1003-9
9406550
J Am Chem Soc. 2006 Sep 6;128(35):11545-50
16939278
Anal Chem. 2001 Jun 1;73(11):2565-70
11403301
Science. 1993 Jun 4;260(5113):1482-6
8502991
Biochemistry. 1993 Sep 21;32(37):9763-71
8396969
Lipids. 1976 Feb;11(2):113-7
814377
J Biol Chem. 1986 Dec 15;261(35):16714-21
3782139
J Am Chem Soc. 2001 Nov 7;123(44):10814-20
11686682
Methods Enzymol. 1993;226:52-71
8277880