Carrie M. Wilmot Associate Professor Oxygen activation in copper amine oxidase University of Michigan April 2008

Carrie M. Wilmot Associate Professor Post-translationally modified amino acid cofactors Okeley & Van der Donk Chemistry & Biology 7, R159- R171 (2000)

Carrie M. Wilmot Associate Professor Roles of Copper Amine Oxidases (CuAOs) Prokaryotes and lower eukaryotes: Amines as N & C sources. Plants: Development, 2 o metabolism. Response to wounding. Animals: Metabolism, regulation of glucose uptake, leukocyte adhesion to vascular cell walls. Increased CuAO activity linked to congestive heart failure, late-diabetic complications and inflammation in humans.

Carrie M. Wilmot Associate Professor Physical properties of CuAOs Homodimeric enzymes monomer kDa ~700 amino acids Contain two cofactors the self-processed cofactor 2,4,5- trihydroxyphenylalanine quinone, or TPQ mononuclear Cu(II) (Type 2 or “non-blue” copper site) 2 4 5

Carrie M. Wilmot Associate Professor Catalytic chemistries: TPQ biosynthesis and amine oxidation R group can be H to proteins Catalyzes the conversion of primary amines to aldehydes

Carrie M. Wilmot Associate Professor Li R. et al. (1998) Structure 6: ; Johnson B.J. et al. (2007) J. Biol. Chem. 282: Hansenula polymorpha CuAO 1.7 Å resolution HPAO

Carrie M. Wilmot Associate Professor Active site of CuAOs Cu-Wa: Å Equatorial Cu ligands: Å Numbering: Hansenula polymorpha CuAO (HPAO)

CuAO catalytic mechanism Reductive half- reaction semiquinone aminoquinol

Carrie M. Wilmot Associate Professor Oxidative half-reaction Substrate reduced enzyme exists in two forms; Aminoquinol / Cu(II)>60% Colorless Semiquinone / Cu(I)<40% Yellow; twin 435, 465nm Following anaerobic amine reduction: Plant CuAOs have ~40% semiquinone / Cu(I) Bacterial CuAOs have ~20% semiquinone / Cu(I) Non-plant eukaryotes ~0% semiquinone / Cu(I)

Carrie M. Wilmot Associate Professor Role of copper in catalysis Postulated mechanism for molecular oxygen reduction in all CuAOs was;

Carrie M. Wilmot Associate Professor Metal replacement studies in yeast HPAO Mills SA, Goto Y, Su Q, Plastino J, & Klinman JP. (2002) Biochemistry 41: Co- and Cu-HPAO have very similar kinetic and chemical mechanisms. Ni(II) and Zn(II) substitutions are inactive. Co-HPAO, where the reduction potential for Co(II)/Co(I) makes Co(I) an unlikely intermediate in catalysis (e.g. –0.4 to –0.5V Co(I)/Co(II) vs SHE in methionine synthase), had a k cat (O 2 ) almost identical to Cu- HPAO under substrate saturating conditions, effectively ruling out the requirement for a Cu redox change during HPAO catalysis. Co(III) was discounted as a kinetically relevant species as the O-18 KIE and k cat (O 2 ) were identical for Cu-HPAO and Co-HPAO. The major difference is a decrease in O 2 affinity in the Co-HPAO. The primary role of the metal is suggested to be electrostatic stabilization of the reduced dioxygen intermediates, and that redox changes at the metal are not required for catalysis.

Carrie M. Wilmot Associate Professor Metal replacement studies in bacterial AGAO Kishishita S, Okajima T, Kim M, Yamaguchi H, Hirota S, Suzuki S, Kuroda S, Tanizawa K, & Mure M. (2003) Role of copper ion in bacterial copper amine oxidase: spectroscopic and crystallographic studies of metal-substituted enzymes. J Am Chem Soc. 125: Co(II)-AGAO (Arthrobacter globiformis) and Ni(II)-AGAO both have k cat (O 2 ) 100-fold down compared to Cu-AGAO. As with Co(II)/Co(I), the reduction potential of Ni(II)/Ni(I) (e.g. 1.16V vs. SHE in complexes with N/O ligands) disfavors a mechanism involving substantial +1 redox character. These studies did not rule out a mechanistic redox role for Cu(I) in the catalytic mechanism of the bacterial enzymes. The lower k cat (O 2 ) for Co-AGAO and Ni-AGAO (1s -1 ) was attributed to molecular oxygen reduction by aminoquinol as the mechanistically relevant species. These k cat (O 2 ) are comparable to the Cu-HPAO and Co-HPAO values (2s -1 ).

Carrie M. Wilmot Associate Professor Model compound 6-amino-4-ethylresorcinol was found to consume oxygen at a rate of 18.6 M -1 s -1 Model compound can activate O 2 6-amino-4-ethylresorcinol Mills SA & Klinman JP (2000) J. Am. Chem. Soc. 122:

CuAO catalytic mechanism off-Cu on-Cu Reductive half- reaction Oxidative half- reaction Bacteria and plants Non-plant eukaryotes

Carrie M. Wilmot Associate Professor Hansenula polymorpha CuAO (HPAO) Yeast enzyme: ―Preferred substrates are small aliphatic primary amines. ―Kinetically most similar to mammalian enzymes (human and bovine plasma). ―Turnover relatively slow, k cat = 3 s -1. ―Among those with low propensity to form measurable semiquinone/Cu(I) upon anaerobic substrate reduction.

Carrie M. Wilmot Associate Professor aminoquinol semiquinone protonated iminoquinone UV/vis spectroscopic features deprotonated iminoquinone TPQ Mure M & Klinman JP (1993) J. Am. Chem. Soc. 115: Hartmann C et al (1993) Biochemistry 32: Mure M & Klinman JP (1995) J. Am. Chem. Soc. 117:

pH 7.0 pH dependence of semiquinone content in HPAO Determined by anaerobic reduction with methylamine. Equilibrium is pulled to semiquinone/Cu(I) by the addition of CN - ions. Δ465 nm is used to quantitate semiquinone fraction. Welford RW, Lam A, Mirica LM & Klinman JP (2007) Biochem. 46: Carrie M. Wilmot Associate Professor

Carrie M. Wilmot Associate Professor Enzymology in crystals [protein] in crystals  [protein] in the cell. The crystal acts likes a porous cage that enables molecules, such as substrates, to diffuse through the solvent channels. Many enzymes retain catalytic activity in the crystal. If there are no large conformational changes during catalysis, many proteins remain crystalline during turnover. Need the majority of the protein molecules in a crystal to be in the same state to “see” that state in the structure. desired intermediate must accumulate must remain stable during X-ray data collection Depending on the system, spectroscopy can track the reaction in the crystalline protein.

Carrie M. Wilmot Associate Professor Single crystal kinetics Hadfield AT & Hajdu J (1993) J. Appl. Cryst. 26: Sjogren T et al. (2002) J. Appl. Cryst. 35:

Carrie M. Wilmot Associate Professor TPQ ~ 480 nm UV/vis spectroscopic features Mure M & Klinman JP (1993) J. Am. Chem. Soc. 115: Hartmann C et al (1993) Biochemistry 32: Mure M & Klinman JP (1995) J. Am. Chem. Soc. 117: Caveat: oxidation state of Cu experimentally undetermined in crystal structures

Oxidized active site 2.6 Å 2.4 Å 2.3 Å TPQ modeled in single conformation (although alternative conformations possible). O5 and D319 C=O separated by intervening water. Room in amine channel for 5 ordered waters. Axial Cu ligand is water (Wa). D319 TPQ Wa Johnson B.J. et al. (2007) J. Biol. Chem. 282:

Trapping intermediates in the crystal Crystal contacts often slow enzyme turnover without changing mechanism. Differential packing may lead to differential subunit reactivity. The asymmetric unit of HPAO contains 3 dimers (6 active sites). 90°

Active sites in the HPAO asymmetric unit 2 : 4 Carrie M. Wilmot Associate Professor Johnson B.J. et al. (2007) J. Biol. Chem. 282:

Methods low oxygen environment

Data collection statistics Carrie M. Wilmot Associate Professor Johnson & Wilmot, unpublished

Carrie M. Wilmot Associate Professor deprotonated iminoquinone ~ 450 nm UV/vis spectroscopic features Mure M & Klinman JP (1993) J. Am. Chem. Soc. 115: Hartmann C et al (1993) Biochemistry 32: Mure M & Klinman JP (1995) J. Am. Chem. Soc. 117:

Methylamine reduction at pH 7.0: the deprotonated iminoquinone Cofactor orientation in all active sites identical to active orientation in oxidized structure. –all cofactors in “off-Cu” conformation. Water structure in amine channel similar to that found in oxidized structure. Diatomic present at axial position of Cu Equatorial bound water is present. 2.4 Å 2.3 Å 2.4 Å

Comparison to E. coli CuAO at steady state ECAO HPAO Wilmot CM et al (1999) Science 286: Carrie M. Wilmot Associate Professor

Carrie M. Wilmot Associate Professor ECAO steady state species Cu Peroxide His526 His Å3.0Å Wilmot CM et al (1999) Science 286: Aerobically trapped steady state species (2.1Å resolution)

Carrie M. Wilmot Associate Professor ECAO steady state species Peroxide Water poised for attack at C5 of iminoquinone to release ammonia. Product aldehyde hydrogen bonded to Asp383, preventing it from ionizing and activating water. Proton transfer pathways to dioxygen; (1) O2 of reduced TPQ dioxygen (2) O4 of reduced TPQ Tyr369 water dioxygen (HPAO: D3)(HPAO: D2)

Carrie M. Wilmot Associate Professor protonated iminoquinone ~ 350 nm UV/vis spectroscopic features Mure M & Klinman JP (1993) J. Am. Chem. Soc. 115: Hartmann C et al (1993) Biochemistry 32: Mure M & Klinman JP (1995) J. Am. Chem. Soc. 117:

Methylamine reduction at pH 6.0: the protonated iminoquinone Direct H-bond between N5 and D319 C=O. Equatorial bound water present. Diatomic present at axial position of Cu. All cofactors in “off- Cu” conformation. 2.8 Å 2.6 Å

Carrie M. Wilmot Associate Professor semiquinone ~ 360, 435 & 465 nm UV/vis spectroscopic features Mure M & Klinman JP (1993) J. Am. Chem. Soc. 115: Hartmann C et al (1993) Biochemistry 32: Mure M & Klinman JP (1995) J. Am. Chem. Soc. 117:

pH dependence of semiquinone content in HPAO Determined by anaerobic reduction with methylamine. Equilibrium is pulled to semiquinone/Cu(I) by the addition of CN - ions. Δ465 nm is used to quantitate semiquinone fraction. Carrie M. Wilmot Associate Professor Welford RW, Lam A, Mirica LM & Klinman JP (2007) Biochem. 46:

Methylamine reduction at pH 8.5: deprotonated iminoquinone /semiquinone mix Cofactor has Cu-on and Cu-off occupancies –50-60% of cofactor directly ligated to Cu Water structure in amine channel consistent with pH 7 deprotonated iminoquinone (450 nm). Equatorial bound water disappears. 2.7 Å 2.3 Å 2.5 Å

Other sites in the asymmetric unit ChainpH 6.0pH 7.0pH 8.5 A B C D E F protonated iminoquinone deprotonated iminoquinone semiquinone

Revisited oxidative half-reaction mechanism favored at high pH favored at low pH acidic basic ? solved in ECAO Carrie M. Wilmot Associate Professor

Summary Elevating pH in methylamine reduced HPAO results in structurally and spectroscopically distinct intermediates; –pH 6.0 protonated iminoquinone –pH 7.0 significant increase in deprotonated iminoquinone –pH 8.5 reveals Cu-on cofactor orientation concurrent with observable semiquinone-Cu(I) species highest k cat in solution for HPAO ~ pH 8.5 Carrie M. Wilmot Associate Professor

Unaddressed questions –Is semiquinone/Cu(I) always copper ligated? –Is a copper ligated semiquinone species reactive with molecular oxygen? –Or is it a non-reactive (off-pathway) species that builds up at high pH? –Does the equatorial site have any role in the oxidative half-reaction? Carrie M. Wilmot Associate Professor

Anaerobic substrate reduced ECAO + azide ions Cu 2+ Azide Aminoquinol (60%) (40%) (60%) His526 His524 His689 Crystal spectrum following X-ray data collection Carrie M. Wilmot Associate Professor Aminoquinol / Cu 2+, with Cu 2+ / azide ion LMCT 380nm 1.9Å resolution

Acknowledgements Wilmot Lab –Dr. Carrie Wilmot –Dr. Bryan Johnson –Dr. Arwen Pearson –Val Klema –Peder Cedervall Klinman Lab (UC Berkeley) –Dr. Judith Klinman –Dr. Richard Welford Beamline Staff (19-ID, Advanced Photon Source) –Dr. Steve Ginell Kahlert Structural Biology Lab –Ed Hoeffner Computer Staff –Dr. Patton Fast Financial Support –National Institutes of Health –Minnesota Medical Foundation –MN Partnership for Biotechnology & Medical Genomics –Minnesota Supercomputing Institute Carrie M. Wilmot Associate Professor Dr. Simon Phillips, Dr. Mike McPherson, Dr. Peter Knowles (Leeds)