title: Reactivity of deoxy- and oxyferrous dehaloperoxidase B from Amphitrite ornata: identification of compound II and its ferrous-hydroperoxide precursor.
abstract: Dehaloperoxidase (DHP) from the terebellid polychaete Amphitrite ornata is a bifunctional enzyme that possesses both hemoglobin and peroxidase activities. The bifunctional nature of DHP as a globin peroxidase appears to be at odds with the traditional starting oxidation state for each individual activity. Namely, reversible oxygen binding is only mediated via a ferrous heme in globins, and peroxidase activity is initiated from ferric centers and to the exclusion of the oxyferrous oxidation state from the peroxidase cycle. Thus, to address what appears to be a paradox, herein we report the details of our investigations into the DHP catalytic cycle when initiated from the deoxy- and oxyferrous states using biochemical assays, stopped-flow UV-visible, and rapid-freeze-quench electron paramagnetic resonance spectroscopies, and anaerobic methods. We demonstrate the formation of Compound II directly from deoxyferrous DHP B upon its reaction with hydrogen peroxide and show that this occurs both in the presence and in the absence of trihalophenol. Prior to the formation of Compound II, we have identified a new species that we have preliminarily attributed to a ferrous-hydroperoxide precursor that undergoes heterolysis to generate the aforementioned ferryl intermediate. Taken together, the results demonstrate that the oxyferrous state in DHP is a peroxidase competent starting species, and an updated catalytic cycle for DHP is proposed in which the ferric oxidation state is not an obligatory starting point for the peroxidase catalytic cycle of dehaloperoxidase. The data presented herein provide a link between the peroxidase and oxygen transport activities, which furthers our understanding of how this bifunctional enzyme is able to unite its two inherent functions in one system.
authors: Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204, USA.
authors: Jennifer
authors: J
authors: D'Antonio
authors: Reza A
authors: RA
authors: Ghiladi
references: Biochemistry. 2008 Dec 9;47(49):12985-94
references: 19006323
references: Arch Biochem Biophys. 2005 Feb 1;434(1):51-9
references: 15629108
references: Acta Crystallogr D Biol Crystallogr. 2007 Oct;63(Pt 10):1094-101
references: 17881827
references: J Phys Chem B. 2005 Feb 24;109(7):3065-70
references: 16851321
references: Biochem Biophys Res Commun. 2007 Nov 30;363(4):954-8
references: 17920039
references: Chembiochem. 2006 Oct;7(10):1582-9
references: 16921577
references: Biochem J. 1997 Aug 15;326 ( Pt 1):109-15
references: 9337857
references: J Biol Chem. 1999 Jan 22;274(4):2029-37
references: 9890961
references: Biochemistry. 2010 Jul 27;49(29):6064-9
references: 20565134
references: J Phys Chem B. 2006 Jul 27;110(29):14483-93
references: 16854160
references: Biochemistry. 2006 Dec 5;45(48):14275-84
references: 17128967
references: Nature. 1999 Sep 30;401(6752):445
references: 10519547
references: J Am Chem Soc. 2010 Dec 15;132(49):17501-10
references: 21090708
references: Acta Crystallogr D Biol Crystallogr. 2010 May;66(Pt 5):529-38
references: 20445228
references: J Biol Chem. 1979 May 10;254(9):3182-6
references: 34616
references: Methods Enzymol. 1994;231:490-6
references: 8041270
references: J Biol Chem. 2005 Mar 11;280(10):9037-42
references: 15637065
references: J Biol Chem. 1984 Nov 10;259(21):13027-36
references: 6092361
references: Eur J Biochem. 1978 Apr 17;85(2):535-47
references: 648533
references: Biochemistry. 2009 May 26;48(20):4231-8
references: 19371065
references: Proteins. 1990;7(4):358-65
references: 2199973
references: Biochemistry. 2008 Nov 4;47(44):11510-7
references: 18844377
references: Nat Struct Biol. 1997 Dec;4(12):1032-8
references: 9406554
references: J Biol Chem. 2007 Sep 28;282(39):28514-28519
references: 17690093
references: Biophys J. 2001 Jul;81(1):435-45
references: 11423426
references: J Biol Chem. 1952 Mar;195(1):133-40
references: 14938361
references: Arch Biochem Biophys. 1988 Aug 1;264(2):438-49
references: 3401009
references: J Biol Chem. 1970 May 10;245(9):2409-13
references: 5442280
references: J Biol Chem. 2005 Dec 9;280(49):40934-8
references: 16221678
references: Methods Enzymol. 1994;233:189-202
references: 8015456
references: Biochemistry. 2009 Feb 10;48(5):995-1005
references: 19187035
references: Proc Natl Acad Sci U S A. 2000 Sep 12;97(19):10354-8
references: 10962034
references: J Biol Chem. 2003 Oct 24;278(43):41597-606
references: 12902339
references: Biochim Biophys Acta. 2008 May;1784(5):863-71
references: 18342641
references: J Biol Chem. 1996 Mar 1;271(9):4609-12
references: 8617721
references: J Am Chem Soc. 2006 Feb 1;128(4):1036-7
references: 16433494
references: Biochemistry. 2006 Aug 1;45(30):9085-94
references: 16866354
references: Biochemistry. 1989 Jul 11;28(14):5714-9
references: 2775733
references: Bioelectrochemistry. 2004 Jun;63(1-2):19-24
references: 15110242
references: Biochem Biophys Res Commun. 2004 Nov 26;324(4):1194-8
references: 15504340
references: J Am Chem Soc. 2008 Feb 20;130(7):2128-9
references: 18217756
references: Biochemistry. 1997 Feb 25;36(8):2197-202
references: 9047320
references: Acta Crystallogr D Biol Crystallogr. 2009 Jan;65(Pt 1):34-40
references: 19153464
references: Biophys J. 1994 Dec;67(6):2236-50
references: 7696465
references: Biochem Biophys Res Commun. 2003 Dec 12;312(2):292-8
references: 14637135
references: Biochim Biophys Acta. 2007 Jan;1774(1):121-30
references: 17182294
references: J Inorg Biochem. 2006 May;100(5-6):1100-8
references: 16603247
references: J Biol Chem. 1999 Jan 29;274(5):2838-44
references: 9915818
references: J Biol Chem. 1978 Aug 25;253(16):5685-9
references: 670221
references: J Biol Chem. 2007 Mar 30;282(13):9646-9656
references: 17202139
references: J Biol Chem. 1991 Dec 15;266(35):23909-15
references: 1748663
references: Biochemistry. 2004 Apr 20;43(15):4601-10
references: 15078107
references: Free Radic Biol Med. 2003 Mar 1;34(5):531-45
references: 12614842
references: Biochemistry. 2010 Aug 10;49(31):6600-16
references: 20545299
references: J Biol Chem. 2000 Jun 23;275(25):18712-6
references: 10751397
references: Biophys J. 2003 Mar;84(3):1998-2004
references: 12609902
references: Biochemistry. 2007 Aug 28;46(34):9823-9
references: 17676875
references: Biochemistry. 2005 Dec 6;44(48):15637-44
references: 16313166
references: J Biol Chem. 2002 Nov 8;277(45):42706-10
references: 12215454
references: FEBS Lett. 1996 Jul 1;389(2):153-6
references: 8766820
references: Biochemistry. 2009 Mar 17;48(10):2164-72
references: 19228049
references: Mar Biotechnol (NY). 2001 May;3(3):287-92
references: 14961367