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Evidence That Dioxygen and Substrate Activation Are Tightly Coupled in Dopamine β-Monooxygenase

2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês

10.1074/jbc.m300797200

1083-351X

John P. Evans, Kyunghye Ahn, Judith P. Klinman,

CO2 Reduction Techniques and Catalysts

Oxygen activation occurs at a wide variety of enzyme active sites. Mechanisms previously proposed for the copper monooxygenase, dopamine β-monooxygenase (DβM), involve the accumulation of an activated oxygen intermediate with the properties of a copper-peroxo or copper-oxo species before substrate activation. These are reminiscent of the mechanism of cytochrome P-450, where a heme iron stabilizes the activated O2 species. Herein, we report two experimental probes of the activated oxygen species in DβM. First, we have synthesized the substrate analog, β,β-difluorophenethylamine, and examined its capacity to induce reoxidation of the prereduced copper sites of DβM upon mixing with O2 under rapid freeze-quench conditions. This experiment fails to give rise to an EPR-detectable copper species, in contrast to a substrate with a C–H active bond. This indicates either that the reoxidation of the enzyme-bound copper sites in the presence of O2 is tightly linked to C-H activation or that a diamagnetic speciesCu(II)-O2· has been formed. In the context of the open and fully solvent-accessible active site for the homologous peptidylglycine-α-hydroxylating monooxygenase and by analogy to cytochrome P-450, the accumulation of a reduced and activated oxygen species in DβM before C-H cleavage would be expected to give some uncoupling of oxygen and substrate consumption. We have, therefore, examined the degree to which O2 and substrate consumption are coupled in DβM using both end point and initial rate experimental protocols. With substrates that differ by more than three orders of magnitude in rate, we fail to detect any uncoupling of O2 uptake from product formation. We conclude that there is no accumulation of an activated form of O2 before C-H abstraction in the DβM and peptidylglycine-α-hydroxylating monooxygenase class of copper monooxygenases, presenting a mechanism in which a diamagnetic Cu(II)-superoxo complex, formed initially at very low levels, abstracts a hydrogen atom from substrate to generate Cu(II)-hydroperoxo and substrate-free radical as intermediates. Subsequent participation of the second copper site per subunit completes the reaction cycle, generating hydroxylated product and water. Oxygen activation occurs at a wide variety of enzyme active sites. Mechanisms previously proposed for the copper monooxygenase, dopamine β-monooxygenase (DβM), involve the accumulation of an activated oxygen intermediate with the properties of a copper-peroxo or copper-oxo species before substrate activation. These are reminiscent of the mechanism of cytochrome P-450, where a heme iron stabilizes the activated O2 species. Herein, we report two experimental probes of the activated oxygen species in DβM. First, we have synthesized the substrate analog, β,β-difluorophenethylamine, and examined its capacity to induce reoxidation of the prereduced copper sites of DβM upon mixing with O2 under rapid freeze-quench conditions. This experiment fails to give rise to an EPR-detectable copper species, in contrast to a substrate with a C–H active bond. This indicates either that the reoxidation of the enzyme-bound copper sites in the presence of O2 is tightly linked to C-H activation or that a diamagnetic speciesCu(II)-O2· has been formed. In the context of the open and fully solvent-accessible active site for the homologous peptidylglycine-α-hydroxylating monooxygenase and by analogy to cytochrome P-450, the accumulation of a reduced and activated oxygen species in DβM before C-H cleavage would be expected to give some uncoupling of oxygen and substrate consumption. We have, therefore, examined the degree to which O2 and substrate consumption are coupled in DβM using both end point and initial rate experimental protocols. With substrates that differ by more than three orders of magnitude in rate, we fail to detect any uncoupling of O2 uptake from product formation. We conclude that there is no accumulation of an activated form of O2 before C-H abstraction in the DβM and peptidylglycine-α-hydroxylating monooxygenase class of copper monooxygenases, presenting a mechanism in which a diamagnetic Cu(II)-superoxo complex, formed initially at very low levels, abstracts a hydrogen atom from substrate to generate Cu(II)-hydroperoxo and substrate-free radical as intermediates. Subsequent participation of the second copper site per subunit completes the reaction cycle, generating hydroxylated product and water. Dopamine β-monooxygenase (DβM) 1The abbreviations used are: DβMdopamine β-monooxygenasePHMpeptidylglycine-β-hydroxylating monooxygenasePHMcccatalytic core of PHMEPRelectron paramagnetic resonanceDFPAβ,β-difluorophenethylamineCuAelectron transfer copperCuBoxygen binding copper: HPLC, high performance liquid chromatographyMES4-morpholineethanesulfonic acid. along with peptidylglycine-α-hydroxylating monooxygenase (PHM) comprise a unique class of enzymes that contain only copper as a cofactor and catalyze the cleavage of O2 to form hydroxylated product and water. DβM is of central importance in the catecholamine biosynthetic pathway, catalyzing the conversion of dopamine to norepinephrine (Scheme 1, top), where both substrate and product serve as neurotransmitters within the central nervous system (1.Stewart L.C. Klinman J.P. Annu. Rev. Biochem. 1988; 57: 551-592Crossref PubMed Google Scholar). Primarily localized within the secretory granules of adrenal chromaffin cells and neurons, DβM is a large, tetrameric glycoprotein (75 kDa per monomer) consisting of two disulfide-linked dimers. dopamine β-monooxygenase peptidylglycine-β-hydroxylating monooxygenase catalytic core of PHM electron paramagnetic resonance β,β-difluorophenethylamine electron transfer copper oxygen binding copper: HPLC, high performance liquid chromatography 4-morpholineethanesulfonic acid. Although no crystal structure has been reported, extensive structural data exist for DβM. Extended X-ray absorption fine structure was used to characterize the ligand environment of the two copper atoms per subunit in both oxidized and reduced forms of DβM; in the absence of any evidence for back scattering between the metal sites, the distance between the two coppers per subunit was concluded to exceed 4 Å (2.Blackburn N.J. Hasnain S.S. Pettingill T.M. Strange R.W. J. Biol. Chem. 1991; 266: 23120-23127Abstract Full Text PDF PubMed Google Scholar, 3.Scott R.A. Sullivan R.J. Dewolf Jr., W.E. Dolle R.E. Kruse L.I. Biochemistry. 1988; 27: 5411-5417Crossref PubMed Scopus (49) Google Scholar). EPR spectroscopy also failed to detect any spin coupling between metal sites in oxidized, resting enzyme (4.Blackburn N.J. Concannon M. Shahiyan S.K. Mabbs F.E. Collison D. Biochemistry. 1988; 27: 6001-6008Crossref PubMed Scopus (31) Google Scholar) and in a catalytically generated product complex (5.Brenner M.C. Murray C.J. Klinman J.P. Biochemistry. 1989; 28: 4656-4664Crossref PubMed Scopus (40) Google Scholar). These findings provided early evidence against a reactive binuclear center and, instead, implicated separate functions for the two copper centers. The CuA (CuH in PHM), liganded by three histidines (DβM: His-255, His-256, His-326; PHM: His-107, His-108, His-172) and water, has historically been assigned to the electron transfer site, and CuB (CuM in PHM), liganded by two histidines (DβM: His-405, His-407; PHM: His-242, His-244) and water together with a long bond to methionine (DβM: Met-480; PHM: Met-314), has historically been assigned to the substrate binding and hydroxylation site. PHM, the second enzyme compromising this class of monooxygenase, catalyzes the first step in the C-terminal amidation of glycine-extended peptides and yields a peptidyl α-hydroxyglycine intermediate (Scheme 1, bottom). This is followed by dealkylation (catalyzed by peptidylglycine-α-amidating lyase) to produce glyoxylate and C-terminal-amidated neuropeptide/hormone. PHM is active as a 35-kDa monomer, designated PHMcc, or as a covalently linked, bifunctional protein with peptidylglycine-α-amidating lyase, designated peptidylglycine-α-amidating monooxygenase (6.Eipper B.A. Stoffers D.A. Mains R.E. Annu. Rev. Neurosci. 1992; 15: 57-85Crossref PubMed Scopus (563) Google Scholar, 7.Prigge S.T. Mains R.E. Eipper B.A. Amzel L.M. Cell. Mol. Life Sci. 2000; 57: 1236-1259Crossref PubMed Scopus (374) Google Scholar). Despite their large difference in size, DβM and PHM share a 28% sequence identity extending through a common catalytic domain of ∼270 residues, which includes the conserved copper ligands (8.Southan C. Kruse L.I. FEBS Lett. 1989; 255: 116-120Crossref PubMed Scopus (68) Google Scholar). In addition both enzymes require two coppers per subunit for full activity (9.Ash D.E. Papadopoulos N.J. Colombo G. Villafranca J.J. J. Biol. Chem. 1984; 259: 3395-3398Abstract Full Text PDF PubMed Google Scholar, 10.Klinman J.P. Krueger M. Brenner M. Edmondson D.E. J. Biol. Chem. 1984; 259: 3399-3402Abstract Full Text PDF PubMed Google Scholar, 11.Kulathila R. Consalvo A.P. Fitzpatrick P.F. Freeman J.C. Snyder L.M. Villafranca J.J. Merkler D.J. Arch. Biochem. Biophys. 1994; 311: 191-195Crossref PubMed Scopus (56) Google Scholar) and are believed to utilize ascorbate as the in vivo two-electron donor (12.Freeman J.C. Villafranca J.J. Merkler D.J. J. Am. Chem. Soc. 1993; 115: 4923-4924Crossref Scopus (49) Google Scholar). The crystal structure of PHM, solved for both oxidized (13.Prigge S.T. Kolhekar A.S. Eipper B.A. Mains R.E. Amzel L.M. Science. 1997; 278: 1300-1305Crossref PubMed Scopus (305) Google Scholar) and reduced (14.Prigge S.T. Kolhekar A.S. Eipper B.A. Mains R.E. Amzel L.M. Nat. Struct. Biol. 1999; 6: 976-983Crossref PubMed Scopus (159) Google Scholar) enzyme, confirmed many of the earlier spectroscopic data. Important features determined from the crystal structure are (i) a two-domain structure in which each domain binds a single copper atom, (ii) a distance of ∼10.6 Å (oxidized PHMcc with bound peptide substrate) between the two copper sites, (iii) the absence of closure of the copper binding domains in either enzyme form studied, and (iv) the identification of a water-filled cavity that is at the solvent interface and “links” the two copper binding domains. Extensive debate has taken place in the recent literature regarding the pathway for electron transfer between copper sites and the nature of O2 activation in DβM and PHM (14.Prigge S.T. Kolhekar A.S. Eipper B.A. Mains R.E. Amzel L.M. Nat. Struct. Biol. 1999; 6: 976-983Crossref PubMed Scopus (159) Google Scholar, 15.Jaron S. Blackburn N.J. Biochemistry. 1999; 38: 15086-15096Crossref PubMed Scopus (70) Google Scholar). Comparison of the kinetic parameters for DβM and PHM with substrates of comparable reactivity indicates the same intrinsic hydrogen/deuterium isotope effect (∼11) for the C-H activation step (16.Francisco W.A. Merkler D.J. Blackburn N.J. Klinman J.P. Biochemistry. 1998; 37: 8244-8252Crossref PubMed Scopus (83) Google Scholar). Additionally, similar O-18 isotope effects for these two enzymes that decrease with substrate deuteration imply a chemical mechanism for substrate oxidation that is likely to be identical (17.Francisco W.A. Blackburn N.J. Klinman J.P. Biochemistry. 2003; 42: 1813-1819Crossref PubMed Scopus (64) Google Scholar). Studies of the kinetic mechanism indicate that both DβM (in the presence of the dianion activator fumarate) and PHM proceed in a preferred ordered mechanism with substrate binding to enzyme before O2 (16.Francisco W.A. Merkler D.J. Blackburn N.J. Klinman J.P. Biochemistry. 1998; 37: 8244-8252Crossref PubMed Scopus (83) Google Scholar, 18.Ahn N. Klinman J.P. Biochemistry. 1983; 22: 3096-3106Crossref PubMed Scopus (71) Google Scholar). Thus, all available data imply that DβM and PHM can be regarded interchangeably with respect to mechanism and active site structure. In early studies of DβM with either substrates or substrate analogs it was concluded that functionalization of substrate involved hydrogen atom abstraction to yield a free radical intermediate (19.Miller S.M. Klinman J.P. Biochemistry. 1985; 24: 2114-2127Crossref PubMed Scopus (116) Google Scholar, 20.Fitzpatrick P.F. Flory Jr., D.R. Villafranca J.J. Biochemistry. 1985; 24: 2108-2114Crossref PubMed Scopus (40) Google Scholar). Identification of the oxygen species catalyzing hydrogen atom abstraction from substrate has proven far more elusive. The observation of pH-dependent isotope effects for DβM provided evidence for the involvement of a single proton in the chemical conversion process, leading to the proposal of a copper hydroperoxide as the reactive oxygen intermediate (III in Scheme 2) (18.Ahn N. Klinman J.P. Biochemistry. 1983; 22: 3096-3106Crossref PubMed Scopus (71) Google Scholar). However, a detailed analysis of the effects of substrate structure and deuteration on O-18 isotope effects (21.Tian G. Berry J.A. Klinman J.P. Biochemistry. 1994; 33: 226-234Crossref PubMed Scopus (118) Google Scholar) was found to be inconsistent with the earlier proposed Cu(II)–OOH 2Note that the charge on oxygen species complexed to copper is omitted when a solid line is drawn between metal and oxygen, e.g. representation of Cu(II)--–OOH (Scheme 2) as Cu(II)–OOH in text. and suggested a reductive cleavage of this intermediate to generate copper-oxo as the hydroxylating agent (IV in Scheme 2). This interpretation assumed classical behavior of hydrogen during transfer from substrate to oxygen, which has now been shown in the case of PHM to be dominated by hydrogen tunneling (22.Francisco W.A. Knapp M.J. Blackburn N.J. Klinman J.P. J. Am. Chem. Soc. 2002; 124: 8194-8195Crossref PubMed Scopus (120) Google Scholar). Additionally, recent site-specific mutagenesis studies with PHM unambiguously eliminates a role for the most plausible active site candidate (PHM: Y318; DβM: Y484) in reductive activation of Cu(II)–OOH (17.Francisco W.A. Blackburn N.J. Klinman J.P. Biochemistry. 2003; 42: 1813-1819Crossref PubMed Scopus (64) Google Scholar). The lack of extensive pH studies for the PHM reaction together with a failure to identify a proton donor from the crystal structure has led to the proposal of mechanism II in Scheme 2 (14.Prigge S.T. Kolhekar A.S. Eipper B.A. Mains R.E. Amzel L.M. Nat. Struct. Biol. 1999; 6: 976-983Crossref PubMed Scopus (159) Google Scholar). A common feature of all previously proposed mechanisms for PHM and DβM(cf. II, III, and IV in Scheme 2) is that oxidation of both copper centers occurs before substrate activation and leads to the accumulation of a partially reduced form of dioxygen. Blackburn et al. (23.Blackburn N.J. Rhames F.C. Ralle M. Jaron S. J. Biol. Inorg. Chem. 2000; 5: 341-353Crossref PubMed Scopus (98) Google Scholar) observe significant changes in the copper coordination structure during oxidation, which would appear to preclude a rapid inter-copper electron transfer. They proposed an alternative possibility based on the finding that CO binds to the electron transfer copper in the presence of peptide substrate; this involves a role for superoxide as the electron carrier between CuA and CuB (15.Jaron S. Blackburn N.J. Biochemistry. 1999; 38: 15086-15096Crossref PubMed Scopus (70) Google Scholar). Many of the paradigms for the copper monooxygenase mechanism have come from the very detailed chemical analyses of cytochrome P-450, which has been shown to release hydrogen peroxide as product either in the absence of a substrate or in the presence of a poor substrate (24.Ortiz de Montellano P.R. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. Plenum Press, New York1986: 217-270Google Scholar). Additionally, activated oxygen at the active site of cytochrome P-450 has been shown to be capable of further reduction to water (25.Kadkhodayan S. Coulter E.D. Maryniak D.M. Bryson T.A. Dawson J.H. J. Biol. Chem. 1995; 270: 28042-28048Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). These side reactions of cytochrome P-450 lead to an uncoupling of substrate hydroxylation from uptake of O2 and occur despite the sequestration of the active site from bulk solvent (26.Poulos T.L. Finzel B.C. Howard A.J. J. Mol. Biol. 1987; 195: 687-700Crossref PubMed Scopus (1295) Google Scholar). For enzymes like DβM and PHM, whose active sites are fully exposed to solvent, it is expected that extensive uncoupling of substrate hydroxylation and O2 uptake may occur. In the case of DβM, the size of the primary isotope effects on kcat and kcat/Km for certain phenethylamine substrates indicates that C-H cleavage is rate-determining, with the implication of a steady state accumulation of an enzyme complex that contains the activated oxygen intermediate. Thus, if the copper monooxygenase mechanism occurs with a build-up of any type of activated oxygen intermediate, we would expect to observe some degree of uncoupling between oxygen reduction and substrate consumption that would increase significantly as the chemical reactivity of the substrate diminishes. The stoichiometry of the DβM (27.Levin E.Y. Levenberg B. Kaufman S. J. Biol. Chem. 1960; 235: 2080-2086Abstract Full Text PDF PubMed Google Scholar) and PHM (28.Murthy A.S.N. Mains R.E. Eipper B.A. J. Biol. Chem. 1986; 261: 1815-1822Abstract Full Text PDF PubMed Google Scholar) reactions with their respective substrates, dopamine and d-Tyr-Val-Gly, was previously shown to be 1 eq of dioxygen consumed for each equivalent of substrate and reductant. We have now extended these studies with DβM using substrates of varying reactivity by changing the para substituent of the phenyl ring. Additionally, single turnover experiments have been performed with a β,β-difluoro analog of phenethylamine that cannot be functionalized. From these results it is possible to rule out the presence of any appreciable level of a reduced oxygen species before substrate functionalization. The extremely tight coupling of O2 and C-H activation in DβM points toward a new mechanistic pathway for DβM and PHM while indicating the inappropriateness of inferences derived from the family of heme-iron-dependent monooxygenases. Materials and General Methods—Soluble DβM was isolated as previously described (5.Brenner M.C. Murray C.J. Klinman J.P. Biochemistry. 1989; 28: 4656-4664Crossref PubMed Scopus (40) Google Scholar) from bovine adrenal glands. The protein concentration was estimated from the absorbance at 280 nm (ϵ280 = 1.24 ml mg–1 cm–1). A monomer mass of 75 kDa was used in calculations of enzyme concentration. Trace metal analysis of enzyme-bound copper was performed on a PerkinElmer 3000DV Inductively Coupled Plasma-Atomic Emission Spectrophotometer using commercially available metal standard solutions. Catalase (65,000 units/mg) was from Roche Applied Science. All other materials were of reagent grade. The compounds 4-hydroxyphenethylamine (tyramine), 3,4-dihydroxyphenethylamine (dopamine), 4-hydroxyphenethanolamine (octopamine), and phenethylamine were purchased from Sigma and used as the hydrochloride salts. [1-14C] tyramine hydrochloride, with a specific activity of 55 mCi/mmol, was purchased from American Radiolabeled Chemicals, Inc. 4-(Trifluoromethyl)phenethylamine hydrochloride was synthesized from the commercially available nitrile (purchased from Aldrich) as previously described (19.Miller S.M. Klinman J.P. Biochemistry. 1985; 24: 2114-2127Crossref PubMed Scopus (116) Google Scholar). The elemental analysis gave 47.91% C, 4.91% H, 6.21% N (calculated: 47.98% C, 5.05% H, 6.05% N). Enzyme Kinetic Assays—Steady-state rates of oxygen consumption were measured with a YSI model 5300 biological oxygen electrode. The assay conditions, similar to those described previously (29.Stewart L.C. Klinman J.P. Biochemistry. 1987; 26: 5302-5309Crossref PubMed Scopus (50) Google Scholar), were as follows: 10 mm disodium fumarate, 10 mm sodium ascorbate, 100 μg/ml catalase, 2 μm CuSO4, 50 mm potassium Pi (pH 6.0), T = 35 °C, 0.218 mm O2 (air saturation). The concentration of the substrates (tyramine, dopamine, and phenethylamine) ranged from 0.05 to 10 mm, whereas the ionic strength was maintained at a constant value of 0.15 m with the addition of NaCl. The concentration of 4-(trifluoromethyl)phenethylamine ranged from 6 to 100 mm, whereas the ionic strength was maintained at 0.2 m with NaCl. The apparent kcat and kcat/Km values were obtained by fitting the data to the Michaelis-Menten equation using the program Kaleidagraph. Enzyme End Point Assays—These were initiated on the oxygen electrode by the addition of various amounts of substrate to a reaction mixture (1 ml) containing 50 mm potassium Pi (pH 6.0), 10 mm disodium fumarate, 10 mm sodium ascorbate, 100 μg/ml catalase, 2 μm CuSO4, and various amounts of DβM. The assays were maintained at a constant temperature of 35 °C using a Neslab circulating water bath. Sufficient amounts of enzyme were added to consume all of the added substrate within 5 min. The concentration of dissolved oxygen under the conditions used was determined to be 218 μm by the protocatechuic acid/protocatechuate dioxygenase assay (30.Wittaker J.W. Orville A.M. Lipscomb J.D. Methods Enzymol. 1990; 188: 82-88Crossref PubMed Scopus (41) Google Scholar). Briefly, the full amplitude of oxygen uptake was recorded after the addition of protocatechuic acid (22.4 μm) to an assay mixture containing 50 mm potassium Pi (pH 6.0), 94 mm NaCl (ionic strength of 0.15 m), and excess of protocatechuate dioxygenase, purchased from Sigma. All end point assays of dopamine, tyramine, and phenethylamine were maintained at an ionic strength of 0.15 m with NaCl. The concentrations of dopamine and tyramine were determined using their reported extinction coefficients: dopamine ϵ280 = 2670 m–1 cm–1, tyramine ϵ274 = 1479 m–1 cm–1. Otherwise, all substrate concentrations were determined by weight. Enzyme Initial Rates Assays—These were performed with the substrates tyramine and 4-(trifluoromethyl)phenethylamine. Assay conditions for tyramine were identical to those described above and were performed on the oxygen electrode. Two tyramine concentrations were used, 5 and 0.2 mm with 0.1 μCi of 14C-labeled tyramine added to trace the extent of reaction. Assays of 4-(trifluoromethyl)phenethylamine contained 50 mm substrate (ionic strength adjusted to 0.2 m with NaCl) and were performed either in the presence or absence of catalase. All assays were quenched with 0.091 m HClO4 (100 μl of a 1 m solution) after 6.5–8.7 min and were immediately cooled to –80 °C and stored for subsequent analysis by HPLC. Total dioxygen consumption was corrected for the ascorbate background rate. All HPLC separations were performed on an Alltech Adsorbosphere reversed phase C-18 column. Octopamine, detected at 224 nm, was separated from tyramine with a mobile phase of 5 mm acetic acid (pH 5.8), 600 μm heptane sulfonic acid, and 15% methanol at 1 ml/min. Separation of the β-hydroxylated product from 4-(trifluoromethyl)phenethylamine was accomplished with an isocratic flow of 17% acetonitrile, 83% water, and 0.1% trifluoroacetic acid at 1 ml/min and was detected at 210 nm. A standard curve based on integrated peak area with mock-quenched reaction mixtures was linear through the range of interest. Blanks, in which no enzyme was added, were used to determine the amount of contaminant eluting with the same retention time as product by HPLC at 0% conversion and were subtracted from each assay. The integrated peak area of this peak corresponded to 5.2–6.0 nmol of product (based on the standard curve constructed from mock quenched assays) in the experiments with catalase present. In those without catalase, the peak integrated to 9.5 nmol of product. The contaminant in the product peak is only 0.01–0.02% of the total amount of 4-(trifluoromethyl)phenethylamine (50 mm) present in the assays. Synthesis of 4-(Trifluoromethyl)phenethanolamine Hydrochloride— 4-(Trifluoromethyl)benzaldehyde was reacted with sodium metabisulfite and sodium cyanide as described previously (31.Levine J. Eble T.E. Fischbach H. J. Am. Chem. Soc. 1948; 60: 1930Crossref Scopus (20) Google Scholar). A saturated solution of sodium metabisulfite (896 mg, 4.7 mmol) was added to 4-(trifluoromethyl)benzaldehyde (1g, 5.7 mmol), and the solution was stirred on ice. A layer of ether (5 ml) was added on top, and an ice-cold saturated solution of sodium cyanide (782 mg, 15.9 mmol) was added. The aqueous layer was diluted with a small amount of water and extracted with ether, and the combined ether solutions were washed with bisulfite solution and then with water. The resulting cyanohydrin was isolated by silica-gel flash chromatography (20% ethyl acetate, 80% hexane) and reduced with LiAlH4 as described in Poos et al. (32.Poos G. Carson J. Rosenau J. Roszkowski A. Kelly N. McGowin J. J. Med. Chem. 1963; 6: 266-272Crossref PubMed Scopus (66) Google Scholar) without further purification. The amine was converted to the hydrochloride salt with HCl in ether. Elemental analysis of the hydrochloride salt was found to contain 44.58% C, 4.29% H, 5.80% N (calculated: 44.74% C, 4.59% H, 5.80% N). Synthesis of β,β-Difluorophenethylamine Hydrochloride—Styrene was first converted to the azirine (2-phenyl-1-azirine) as described previously (33.Hortmann A.G. Robertson D.A. Gillard B.K. J. Org. Chem. 1972; 37: 322-324Crossref Scopus (95) Google Scholar). To a solution of styrene (52.19 g, 57.5 ml, 0.50 mol) in 400 ml of CCl4 in an ice bath was added a solution of bromine (80 g, 25.8 ml, 0.50 mol) in 100 ml of CCl4 through an addition funnel over 30 min. The reaction mixture was stirred at room temperature for 1 h. The solvent was evaporated under vacuum to yield 1,2-dibromo-2-phenyl-benzene, a white solid that was immediately dissolved in 750 ml of dry Me2SO and placed in a 1-liter 3-necked round-bottomed flask. While on an ice bath NaN3 was added (49 g, 0.75 mol) with stirring and under nitrogen. After 12 h the reaction mixture was cooled on an ice bath, and 20 g of NaOH (0.5 mol) in 20 ml of H2O was added. After 1 h of stirring only one spot was visible by TLC (n-hexane). The mixture was diluted to a volume of 2 liters with 2% NaHCO3 in H2O and extracted with methylene chloride 4 × 200 ml, and the combined extracts were washed with H2O, filtered, and concentrated in vacuo to yield a dark brown liquid. This was diluted with petroleum ether (200 ml) and applied to an alumina (basic, 200 g) column with petroleum ether as the eluent. This gave 72.8 g of a light yellow oil, which was dissolved in 1.25 liter of toluene. 1.2 liters of the α-azidostyrene in toluene was placed in a round-bottom flask. The mixture was heated under reflux, and progress of the reaction was monitored by TLC (9:1, n-hexanes:ethyl acetate). After 1 h and 20 min the reaction was stopped. The product was distilled to yield 37.02 g of 2-phenyl-1-azirine, a colorless oil. Next, the azirine was converted to the β,β-difluoroamine by reaction with HF in pyridine (34.Wade T.N. Kheribet R. J. Org. Chem. 1980; 45: 5333-5335Crossref Scopus (26) Google Scholar). 12 g (0.1 mol) of 2-phenyl-1-azirine was added to 120 ml of 70% HF in pyridine at –35 to –30 °C over 15 min and stirred at –25 to 0 °C for 30 min and for 80 min at room temperature. The reaction was quenched with the addition of 60 ml of water at –78 °C. Finally the amine was converted to the hydrochloride salt by heating in an HCl solution under reflux for 0.5 h. The solvent was evaporated and solid-washed with acetone. The white solid was recrystallized 3 times from a mixture of ethanol and ether to give 1.26 g (7% yield) of the hydrochloride salt. The final product was found to have a melting point of 181–183 °C, and the elemental analysis gave 49.50% C, 5.15% H, 7.35% N (calculated: 49.63% C, 5.21% H, 7.23% N). Freeze-quench Experiments with DβM—A detailed description of the set-up as well as the experimental procedures can be found in Brenner et al. (5.Brenner M.C. Murray C.J. Klinman J.P. Biochemistry. 1989; 28: 4656-4664Crossref PubMed Scopus (40) Google Scholar). A dead time of 4 ms was estimated as described previously. The samples used for EPR analysis were quenched at various time points ranging from 9 to 470 ms. The manner in which reaction solutions were mixed for a sample with tyramine was the following; initially, equal volumes of the enzyme solution consisting of 60 μm DβM monomer, 100 mm potassium Pi (pH 6.19), 30.6 μm endogenous copper, 89.4 μm exogenous CuSO4, 190 mm KCl, and 20 mm fumarate was mixed with an equal volume of a 60 μm ascorbic acid, 10 mm HCl solution. This reduced enzyme solution was then reacted with an equal volume of substrate stock solution, 10 mm tyramine, 90 mm KCl, 10 mm fumarate, and 50 mm potassium Pi (pH 6.0) to yield the final conditions. These contained 15 μm DβM (2 Cu2+/subunit), 100 mm total chloride adjusted with KCl, 15 μm ascorbate, 10 mm fumarate, 50 mm potassium Pi (pH 6.0), 30 μm Cu2+, and either 5 mm tyramine or 40 mm β,β-difluorophenethylamine (DFPA) at 25 °C. Air oxidation of ascorbate-reduced enzyme was studied under identical conditions as described above but lacking tyramine or DFPA substrate. Electron Paramagnetic Measurements—EPR measurements of the Cu(II) content of the enzyme were performed as described previously (5.Brenner M.C. Murray C.J. Klinman J.P. Biochemistry. 1989; 28: 4656-4664Crossref PubMed Scopus (40) Google Scholar). The following settings were used: 5 milliwatts microwave power, 20-G modulation amplitude, 2970 ± 500-G field sweep, sweep time 57 s/scan × 4 scans, 0.3-s time constant, and T ≅ 15 K. Determination of Ki for DFPA—To test the inhibitory effect of DFPA, oxygen electrode assays were performed in the presence of 50 mm MES (pH 6.0), 10 mm fumarate, 100 μg/ml catalase, 10 mm ascorbate, 2 μm CuSO4, and 10 μg/ml DβM at 35 °C. The chloride ion concentration was kept constant at 110 mm with the addition of KCl. The concen