Isocyanide binding to the copper(I) centers of the catalytic core of peptidylglycine monooxygenase (PHMcc).

Binding of the Cu(I)-specific ligands 2,6-dimethylphenyl isocyanide (DIMPI) and isopropyl isocyanide (IPI) to the reduced form of peptidylglycine monooxygenase (PHM) is reported. Both ligands bind to the methionine-containing CuM center, eliciting FTIR bands at 2,138 and 2,174 cm(-1), respectively, but appear unable to coordinate at the histidine-containing CuH center in the wild-type enzyme. This chemistry parallels that previously observed for CO binding to the reduced PHM catalytic core (PHMcc). However, in contrast to the CO chemistry, peptide substrate binding did not induce binding of the isocyanide at CuH. XAS confirmed the binding of DIMPI at CuM via the observation of a short Cu-C interaction at 1.87 A and by the lengthening of the Cu-S(methionine) bond length by 0.06 A. Similarly, FTIR studies on DIMPI binding to the M314I and H172A mutant forms of reduced PHMcc confirmed the assignment of the 2,138-cm(-1) IR band as a CuM-DIMPI complex, but surprisingly also showed DIMPI binding to CuH, as indicated by a band at 2,148 cm(-1). An inorganic complex, [Cu(1,2-Me2Im)2(DIMPI)](PF6), was synthesized and its crystal structure was determined as a model for the interaction of isocyanides with imidazole-containing Cu(I) complexes. Comparison of EXAFS data for the protein and model suggests that DIMPI probably binds to CuM in a tilted fashion, similar to that of ethyl isocyanide binding to myoglobin.

Characterization of a half-apo derivative of peptidylglycine monooxygenase. Insight into the reactivity of each active site copper.

A derivative of peptidylglycine monooxygenase which lacks the CuH center has been prepared and characterized. This form of the enzyme is termed the half-apo protein. Copper-to-protein stoichiometric measurements establish that the protein binds only one of the two copper centers (CuM and CuH) found in the native enzyme. Confirmation that the methionine-containing CuM has been retained has been obtained from EXAFS experiments which show that the characteristic signature of the Cu-S(Met) interaction is preserved. The half-apo derivative binds 1 equiv of CO per copper with an IR frequency of 2092 cm(-1), and this monocarbonyl also displays the Cu-S(Met) interaction in its EXAFS spectrum. These results allow unambiguous assignment of the 2092 cm(-1) band as a CuM-CO species. Binding of CO in the presence of peptide substrate was also investigated. In the native enzyme, substrate induced binding of a second CO molecule with an IR frequency of 2062 cm(-1), tentatively assigned to a CO complex of the histidine-containing CuH site. Unexpectedly, this reactivity is also observed in the half-apo derivative, although the intensity distribution of the CO stretches now indicates that the copper has been partially transferred to a second site, believed to be CuH. The implications of this observation are discussed in terms of a possible additional peptide binding site close to the CuH center.

Major changes in copper coordination accompany reduction of peptidylglycine monooxygenase: implications for electron transfer and the catalytic mechanism.

X-ray absorption spectroscopy has been used to probe the local coordination of the copper centers in the oxidized and reduced states of the peptidylglycine monooxygenase catalytic core (PHMcc) in both the resting and substrate-bound forms of the enzyme. The results indicate that reduction causes significant changes in coordination number and geometry of both Cu centers (CuH and CuM). The CuH center changes from 4- or 5-coordinate tetragonal to a 2-coordinate configuration, with one of the three histidine ligands becoming undetectable by EXAFS (suggesting that it has moved away from the CuH by at least 0.3 A). The CuM center changes from 4- or 5-coordinate tetragonal to a trigonal or tetrahedral configuration, with an estimated 0.3-0.5 A movement of the M314 S ligand. Reduction also leads to loss of coordinated water from both of the coppers. Substrate binding has little or no effect on the local environment of the Cu centers in either oxidation state. These findings bring into question whether direct electron transfer between CuH and CuM via a tunneling mechanism can be fast enough to support the observed catalytic rate, and suggest that some other mechanism for electron transfer, such as superoxide channeling, should be considered.

Domains I and III of the human copper chaperone for superoxide dismutase interact via a cysteine-bridged Dicopper(I) cluster.

Copper binding to the human copper chaperone for superoxide dismutase (hCCS) has been investigated by X-ray absorption spectroscopy. Stoichiometry measurements on the dialyzed, as-isolated protein indicated that up to 3.5 Cu ions bound per hCCS molecule. Reduction with either sodium dithionite or dithiothreitol decreased the copper binding ratio to 2 coppers per hCCS monomer. Analysis of the as-isolated EXAFS data indicated coordination of Cu by a mixture of S and N backscatterers, suggestive of heterogeneous binding of copper between Cu-cysteine binding sites of domain I or III and copper-histidine SOD1-like metal binding sites of domain II. The best fit was obtained with 1.6 Cu-S (cysteine) at 2.24 A (2sigma(2) = 0.011 A(2)) and 1.1 N (histidine) at 1.98 A (2sigma(2) = 0.005 A(2)). A peak of variable intensity in the Fourier transform (FT) of the as-isolated protein at 2.7 A was suggestive of the presence of a heavy atom scatterer such as Cu. Analysis of the dithionite- and DTT-reduced derivatives indicated that copper was lost from the histidine coordinating sites, resulting in a S-only environment with copper coordinated to three S backscatterers at 2. 26 A. The heavy atom scatterer peak was now prominent in the FT and could be well fit by a Cu-Cu interaction at 2.72 A. The data were best interpreted by a dinuclear mu(2)()-bridged cluster with doubly bridging cysteine ligands similar to the cluster proposed to exist in the cytochrome c oxidase chaperone COX17. Analysis of primary sequence and X-ray structural information on yeast CCS strongly suggests that this cluster bridges between domains I and III in hCCS. A mechanism for copper translocation is briefly discussed.

Spectroscopic analysis of the trinuclear cluster in the Fet3 protein from yeast, a multinuclear copper oxidase.

The Fet3 protein (Fet3p) is a multinuclear copper oxidase essential for high-affinity iron uptake in yeast. Fet3p contains one type 1, one type 2, and a strongly antiferromagnetically coupled binuclear Cu(II)-Cu(II) type 3 copper. The type 2 and type 3 sites constitute a structurally distinct trinuclear cluster at which dioxygen is reduced to water. In Fet3p, as in ceruloplasmin, Fe(II) is oxidized to Fe(III) at the type 1 copper; this is the ferroxidase reaction that is fundamental to the physiologic function of these two enzymes. Using site-directed mutagenesis, we have generated type 1-depleted (T1D), type 2-depleted (T2D), and T1D/T2D mutants. None were active in the essential ferroxidase reaction catalyzed by Fet3p. However, the spectroscopic signatures of the remaining Cu(II) sites in any one of the three mutants were indistinguishable from those exhibited by the wild type. Although the native protein and the T1D mutant were isolated in the completely oxidized Cu(II) form, the T2D and T1D/T2D mutants were found to be completely reduced. This result is consistent with the essential role of the type 2 copper in dioxygen turnover, and with the suggestions that cuprous ion is the valence state of intracellular copper. Although stable to dioxygen, the Cu(I) sites in both proteins were readily oxidized by hydrogen peroxide. The double mutant was extensively analyzed by X-ray absorption spectroscopy. Edge and near-edge features clearly distinguished the oxidized from the reduced form of the binuclear cluster. EXAFS was strongly consistent with the expected coordination of each type 3 copper by three histidine imidazoles. Also, copper scattering was observed in the oxidized cluster along with scattering from a ligand corresponding to a bridging oxygen. The data derived from the reduced cluster indicated that the bridge was absent in this redox state. In the reduced form of the double mutant, an N/O ligand was apparent that was not seen in the reduced form of the T1D protein. This ligand in T1D/T2D could be either the remaining type 2 copper imidazole ligand (from His416) or a water molecule that could be stabilized at the type 3 cluster by H-bonding to this side chain. If present in the native protein, this H(2)O could provide acid catalysis of dioxygen reduction at the reduced trinuclear center.

Does superoxide channel between the copper centers in peptidylglycine monooxygenase? A new mechanism based on carbon monoxide reactivity.

Peptidylglycine monooxygenase (PHM) carries out the hydroxylation of the alpha-C atom of glycine-extended propeptides, the first step in the amidation of peptide hormones by the bifunctional enzyme peptidyl-alpha-amidating monooxygenase (PAM). Since PHM is a copper-containing monooxygenase, a study of the interaction between the reduced enzyme and carbon monoxide has been carried out as a probe of the interaction of the Cu(I) sites with O(2). The results show that, in the absence of peptide substrate, reduced PHM binds CO with a stoichiometry of 0.5 CO/Cu(I), indicating that only one of the two copper centers, Cu(B), forms a Cu(I)-carbonyl. FTIR spectroscopy shows a single band in the 2200-1950 cm(-)(1) energy region with nu(CO) = 2093 cm(-)(1) assigned to the intraligand C-O stretch via isotopic labeling with (13)CO. A His242Ala mutant of PHM, which deletes the Cu(B) site by replacing one of its histidine ligands, completely eliminates CO binding. EXAFS spectroscopy is consistent with binding of a single CO ligand with a Cu-C distance of 1.82 +/- 0.03 A. The Cu-S(met) distance increases from 2.23 +/- 0. 02 A in the reduced unliganded enzyme to 2.33 +/- 0.01 A in the carbonylated enzyme, suggesting that the methionine-containing Cu(B) center is the site of CO binding. The binding of the peptide substrate N-Ac-tyr-val-gly perturbs the CO ligand environment, eliciting an IR band at 2062 cm(-)(1) in addition to the 2093 cm(-)(1) band. (13)CO isotopic substitution assigns both frequencies as C-O stretching bands. The CO:Cu binding stoichiometry and peptide/CO FTIR titrations indicate that the 2062 cm(-)(1) band is due to binding of CO at a second site, most likely at the Cu(A) center. This suggests that peptide binding may activate the Cu(A) center toward O(2) binding and reduction to superoxide. As a result of these findings, a new mechanism is proposed involving channeling of superoxide across the 11 A distance between the two copper centers.

Selenomethionine-substituted Thermus thermophilus cytochrome ba3: characterization of the CuA site by Se and Cu K-EXAFS.

We have designed a gene that encodes a polypeptide corresponding to amino acids 44-168 of the Thermus thermophilus cytochrome ba3 subunit II [Keightley et al. (1995) J. Biol. Chem. 270, 20345-20358]. The resulting ba3-CuAt10 protein separated into two fractions (A and B) during cation exchange chromatography which were demonstrated to differ only by N-terminal acetylation in fraction A. When the gene was expressed in an Escherichia coli strain that is auxotrophic for methionine and grown in the presence of selenomethionine (Se(Met)), the single methionine of the CuAt10 protein was quantitatively replaced with Se(Met). Native (S(Met)) and Se(Met)-substituted proteins were characterized by electrospray mass, optical absorption, and EPR spectroscopies and by electrochemical analysis; they were found to have substantially identical properties. The Se(Met)-containing protein was further characterized by Se and Cu K-EXAFS which revealed Cu-Se bond lengths of 2.55 A in the mixed-valence form and 2.52 A in the fully reduced form of CuA. Further analysis of the Se- and Cu-EXAFS spectra yielded the Se-S(thiolate) distances and thereby information on the Se-Cu-Cu and Se-Cu-S(thiolate) angles. An expanded EXAFS structural model is presented.

Coordination of CuB in reduced and CO-liganded states of cytochrome bo3 from Escherichia coli. Is chloride ion a cofactor?

The ubiquinol oxidase cytochrome bo3 from Escherichia coli is one of the respiratory heme-copper oxidases which catalyze the reduction of O2 to water linked to translocation of protons across the bacterial or mitochondrial membrane. We have studied the structure of the CuB site in the binuclear heme-copper center of O2 reduction by EXAFS spectroscopy in the fully reduced state of this enzyme, as well as in the reduced CO-liganded states where CO is bound either to the heme iron or to CuB. We find that, in the reduced enzyme, CuB is coordinated by one weakly bound and two strongly bound histidine imidazoles at Cu-N distances of 2.10 and 1.92 A, respectively, and that an additional feature at 2.54 A is due to a highly ordered water molecule that might be weakly associated with the copper. Unexpectedly, the binding of CO to heme iron is found to result in a major conformational change at CuB, which now binds only two equidistant histidine imidazoles at 1.95 A and a chloride ion at 2. 25 A, with elimination of the water molecule and one of the histidines. Attempts to remove the chloride from the enzyme by extensive dialysis did not change this finding, nor did substitution of chloride with bromide. Photolysis of CO bound to the heme iron is known to cause the CO to bind to CuB in a very fast reaction and to remain bound to CuB at low temperatures. In this state, we indeed find the CO to be bound to CuB at a Cu-C distance of 1.85 A, with chloride still bound at 2.25 A and the two histidine imidazoles at a Cu-N distance of 2.01 A. These results suggest that reduction of the binuclear site weakens the bond between CuB and one of its three histidine imidazole ligands, and that binding of CO to the reduced binuclear site causes a major structural change in CuB in which one histidine ligand is lost and replaced by a chloride ion. Whether chloride is a cofactor in this enzyme is discussed.

The CuA domain of Thermus thermophilus ba3-type cytochrome c oxidase at 1.6 A resolution.

The structure of the CuA-containing, extracellular domain of Thermus thermophilus ba3-type cytochrome c oxidase has been determined to 1.6 A resolution using multiple X-ray wavelength anomalous dispersion (MAD). The Cu2S2 cluster forms a planar rhombus with a copper-copper distance of 2.51 +/- 0.03 A. X-ray absorption fine-structure (EXAFS) studies show that this distance is unchanged by crystallization. The CuA center is asymmetrical; one copper is tetrahedrally coordinated to two bridging cysteine thiolates, one histidine nitrogen and one methionine sulfur, while the other is trigonally coordinated by the two cysteine thiolates and a histidine nitrogen. Combined sequence-structure alignment of amino acid sequences reveals conserved interactions between cytochrome c oxidase subunits I and II.

Kinetic mechanism and intrinsic isotope effects for the peptidylglycine alpha-amidating enzyme reaction.

The bifunctional peptidylglycine alpha-amidating enzyme catalyzes the C-terminal amidation of glycine-extended peptides. The first enzyme activity, peptidylglycine alpha-hydroxylating monooxygenase, catalyzes the oxygen-, ascorbate-, and copper-dependent formation of alpha-hydroxyglycine derivatives. These are substrates for the second enzyme activity, peptidylamidoglycolate lyase, which catalyzes their breakdown to the corresponding C-terminal amidated peptide and glyoxylate as final products. Kinetic and isotope effect studies were carried out with N-benzoylglycine as a substrate at pH 6.0 using monofunctional and bifunctional monooxygenase activities. Kinetic data indicate an equilibrium ordered mechanism, with hippuric acid binding first followed by oxygen. A potentially important difference between the two monooxygenase activities is that product release occurs more slowly from the bifunctional enzyme, indicating an influence of the lyase domain on release of alpha-hydroxyglycine product to solution. Intrinsic isotope effects for the C-H bond cleavage were measured for the monofunctional form of the enzyme using a double-label tracer method, yielding 10.6 +/- 0.8 and 1.20 +/- 0.03 for the primary and alpha-secondary deuterium intrinsic isotope effects, respectively. These values are identical to previous measurements for the analogous enzyme system, dopamine beta-monooxygenase [Miller, S. M., and Klinman, J. P. (1985) Biochemistry 24, 2114-2127]. The identity of intrinsic isotope effects for peptidylglycine alpha-hydroxylating monooxygenase and dopamine beta-monooxygenase with substrates of comparable reactivity (N-benzoylglycine and dopamine, respectively) extends similarities between the two enzymes significantly beyond sequence homology and cofactor requirements.

Purification and characterization of laccases from the white-rot basidiomycete Dichomitus squalens.

Two chromatographic forms of laccase c1 and c2 were purified approximately 225-fold from the extracellular culture fluid of ligninolytic cultures of Dichomitus squalens, using DEAE-Sepharose and Mono-Q fast protein liquid chromatography. Each homogeneous laccase (c1 and c2) has a molecular mass of approximately 66 kDa as determined by SDS-PAGE. Both forms are glycoproteins, and each contains four copper atoms per molecule of protein. The first 20 amino acids of the N-terminal sequences of these two laccases are identical and are similar to those of laccases from other lignin-degrading fungi. The electronic absorption spectra of these laccases exhibit bands at 610 and 330 nm, indicative of type I and type III copper. The EPR spectrum of laccase c1 exhibits bands indicative of type I and type II copper. Each laccase oxidizes a variety of phenolic substrates, has a pH optimum of 3.0 for the oxidation of 2,6-dimethoxyphenol, and is inhibited strongly by fluoride and azide.

Structural and functional investigations on the role of zinc in bifunctional rat peptidylglycine alpha-amidating enzyme.

Bifunctional peptidylglycine alpha-amidating enzyme (alpha-AE) catalyzes the two-step conversion of C-terminal glycine-extended peptides to C-terminal alpha-amidated peptides and glyoxylate. The first step is the ascorbate-, O2-, and copper-dependent hydroxylation of the alpha-carbon of the glycyl residue, producing an alpha-hydroxyglycine-extended peptide. The second step is the ascorbate-, O2-, and copper-independent dealkylation of the carbinolamide intermediate. We show that alpha-AE requires 1.1 +/- 0. 2 mol of zinc/mol of enzyme for maximal (S)-N-dansyl-Tyr-Val-alpha-hydroxyglycine dealkylation activity. Treatment of the enzyme with EDTA abolishes both the peptide hydroxylation and the carbinolamide dealkylation activities. Addition of Zn(II), Co(II), Cd(II), and Mn(II) partially restores carbinolamide dealkylation activity to the EDTA-treated enzyme. Addition of Co(II) produces the greatest restoration of dealkylation activity, 32% relative to a control not treated with EDTA, while Mn(II) addition results in the smallest restoration of dealkylation activity, only 3% relative to an untreated control. The structure and coordination of the zinc center has been investigated by X-ray absorption spectroscopy. EXAFS data are best interpreted by an average coordination of 2-3 histidine ligands and 1-2 non-histidine O/N ligands. Since catalytic zinc centers in other zinc metalloenzymes generally exhibit only O/N ligands to the zinc atom, a zinc-bound water or hydroxide may serve as a general base for the abstraction of the hydroxyl proton from the carbinolamide intermediate. Alternatively, the zinc may function in a structural role.

Structural investigations on the coordination environment of the active-site copper centers of recombinant bifunctional peptidylglycine alpha-amidating enzyme.

The structure and coordination chemistry of the copper centers in the bifunctional peptidylglycine alpha-amidating enzyme (alpha-AE) have been investigated by EPR, EXAFS, and FTIR spectroscopy of a carbonyl derivative. The enzyme contains 2 coppers per 75 kDa protein molecule. Double integration of the EPR spectrum of the oxidized enzyme indicates that 98 +/- 13% of the copper is EPR detectable, indicating that the copper centers are located in mononuclear coordination environments. The Cu(II) coordination of the oxidized enzyme is typical of type 2 copper proteins. EXAFS data are best interpreted by an average coordination of 2-3 histidines and 1-2 O/N (probably O from solvent, Asp or Glu) as equatorial ligands. Reduction causes a major structural change. The Cu(I) centers are shown to be structurally inequivalent since only one of them binds CO. EXAFS analysis of the reduced enzyme data indicates that the nonhistidine O/N shell is displaced, and the Cu(I) coordination involves a maximum of 2.5 His ligands together with 0.5 S/CI ligand per copper. The value of v(CO) (2093 cm-1) derived from FTIR spectroscopy suggests coordination of a weak donor such as methionine, which is supported by a previous observation that the delta Pro-PHM382s mutant M314I is totally inactive. Binding of the peptide substrate N-Ac-Tyr-Val-Gly causes minimum structural perturbation at the Cu(I) centers but appears to induce a more rigid conformation in the vicinity of the S-Met ligand. The unusually intense 8983 eV Cu K-absorption edge feature in reduced and substrate-bound-reduced enzymes is suggestive of a trigonal or digonal coordination environment for Cu(I). A structural model is proposed for the copper centers involving 3 histidines as ligands to CuIA and 2 histidines and 1 methionine as ligands to CuIB. However, in view of the intense 8934 eV edge feature and the lack of CO-binding ability, a 2-coordinate structure for CuA is also entirely consistent with the data.

Structure of CuB in the binuclear heme-copper center of the cytochrome aa3-type quinol oxidase from Bacillus subtilis: an ENDOR and EXAFS study.

We have studied the structure of the CuB site in the binuclear heme-copper center of the fully oxidized form of the quinol-oxidizing cytochrome aa3-600 from Bacillus subtilis by EXAFS and ENDOR spectroscopy. This enzyme is member of the large superfamily of heme-copper respiratory oxidases, which catalyze the reduction of dioxygen to water and link it to translocation of protons across the bacterial or mitochondrial membrane. The EXAFS of the CuB site strongly suggests tetragonal coordination by two or three histidines with one or two O/N donor ligands. There are some indications that a Cl- ion might fractionally occupy substitution-labile sites, although the majority of enzyme molecules did not contain any heavy (second row) scatters, indicative of a Cl- (or S) bridge between the heme iron and CuB [cf. Powers, L., et al. (1994) Biochim. Biophys. Acta 1183, 504-512]. Proton ENDOR spectroscopy of the CuB site in 1H2O and 2H2O media showed evidence of an oxygenous copper ligand with an exchangeable proton. 14N ENDOR revealed three inequivalent nitrogenous ligands with hyperfine coupling constants consistent with histidines. Together, these results strongly suggest that the fully oxidized enzyme has a low-symmetry, tetragonal CuB site with three histidine nitrogens and one oxygen as ligands, the latter with an exchangeable proton(s). The identity and assignment of these ligands are discussed.

The catalytic core of peptidylglycine alpha-hydroxylating monooxygenase: investigation by site-directed mutagenesis, Cu X-ray absorption spectroscopy, and electron paramagnetic resonance.

Peptidylglycine alpha-hydroxylating monooxygenase (PHM) is a copper, ascorbate, and molecular oxygen dependent enzyme that plays a key role in the biosynthesis of many peptides. Using site-directed mutagenesis, the catalytic core of PHM was found not to extend beyond Asp359. Shorter PHM proteins were eliminated intracellularly, suggesting that they failed to fold correctly. A set of mutant PHM proteins whose design was based on the structural and mechanistic similarities of PHM and dopamine beta-monooxygenase (D beta M) was characterized. Mutation of Tyr79, the residue equivalent to a p-cresol target in D beta M, to Phe79 altered the kinetic parameters of PHM. Disruption of either His-rich cluster contained within the PHM/D beta M homology domain eliminated activity, while deletion of a third His-rich cluster unique to PHM failed to affect activity; the catalytically inactive mutant PHM proteins still bound to a peptidylglycine substrate affinity resin. EPR and EXAFS studies of oxidized PHM indicate that the active site contains type 2 copper in a tetragonal environment; the copper is coordinated to two to three His and one to two additional O/N ligands, probably solvent, again supporting the structural homology of PHM and D beta M. Mutation of the Met residues common to PHM and D beta M to Ile identified Met314 as critical for catalytic activity.

Metal-metal bonding in biology: EXAFS evidence for a 2.5 A copper-copper bond in the CuA center of cytochrome oxidase.

Evidence for a direct Cu-Cu bond in the CuA center of cytochrome oxidase is reported. Simulation of the X-ray absorption spectrum of a recombinant CuA-binding domain of Bacillus subtilis cytochrome oxidase, and comparison with a structurally characterized directly-bonding Cu(1.5) ... Cu(1.5) inorganic complex, suggests that a Cu-Cu interaction of 2.5 +/- 0.1 A together with a short 2.2 A Cu-S interaction may be present in the CuA site. In light of these data, previous interpretations of the EXAFS of a number of cytochrome oxidase and nitrous oxide reductase enzymes which modeled the 2.6 A interaction as a long Cu-S(methionine) bond are possibly incorrect. A structural model based on the new data is presented which suggests that the CuA sites in cytochrome oxidase and N2O reductase are likely composed of a pair of modified type 1 copper centers with one histidine, one cysteine, and one weakly bound ligand (Met and/or Gln) joined by a Cu-Cu bond.

X-ray absorption studies of the Cu-dependent phenylalanine hydroxylase from Chromobacterium violaceum. Comparison of the copper coordination in oxidized and dithionite-reduced enzymes.

The coordination chemistry of the Cu sites of phenylalanine hydroxylase (PAH) from Chromobacterium violaceum has been studied by X-ray absorption spectroscopy (XAS). The EXAFS of the Cu(II) form of the enzyme resembles that of other non-blue copper proteins such as plasma amine oxidases and dopamine-beta-hydroxylase and is characteristic of a mixed N/O coordination shell containing histidine ligation. Detailed simulations of the raw EXAFS data have been carried out using full curved-wave restrained refinement methodologies which allow imidazole ligands to be treated as structural units. The results suggest a Cu(II) coordination of two histidines and two additional O/N-donor groups. A reasonable fit to both data sets can be obtained by assuming that the non-imidazole first-shell donor atoms are derived from solvent (H2O or OH-). The EXAFS of the reduced enzyme shows major differences. The amplitude of the first shell in the Fourier transform is only 50% of that of the oxidized enzyme, indicative of a substantial reduction in coordination number. In addition, the first shell of the transform is split into two components. Simulations of the reduced data can be obtained by either two histidines at a long distance of 2.08 A and an O ligand at a short distance of 1.88 A or two histidines at a short distance of 1.90 A and one second-row scatterer such as S or Cl at 2.20 A. Comparison of absorption edge data on the reduced enzyme with data from Cu(I) bis- and tris(1,2-dimethylimidazole) complexes suggests a pseudo-three-coordinate structure.

Copper K-extended x-ray absorption fine structure studies of oxidized and reduced dopamine beta-hydroxylase. Confirmation of a sulfur ligand to copper(I) in the reduced enzyme.

The structure of the copper sites in oxidized and reduced dopamine beta-hydroxylase has been studied by extended x-ray absorption fine structure spectroscopy using a restrained refinement approach to data analysis. An histidine-rich active site has been found to be present with an average histidine coordination of between two and three histidine ligands per copper. In the oxidized protein, the data support four-coordination, involving two to three imidazole groups at 1.99 A with additional ligands derived from water or exogenous O-donor groups at an average distance of 1.94 A. Studies on the reduced enzyme have focused on resolving the controversy in the literature (Scott, R. A., Sullivan, R. J., De Wolfe, W. E., Dolle, R. E., and Kruse, L. I. (1988) Biochemistry 27, 5411-5417; Blumberg, W. E., Desai, P. R., Powers, L., Freedman, J. H., and Villafranca, J. J. (1989) J. Biol. Chem. 264, 6029-6032) as to whether a S/Cl scatterer is a ligand to Cu(I). Five independent samples of reduced enzyme prepared under conditions designed to probe the Cu(I) ligand environment have been measured and analyzed. All five samples gave identical spectra and could be simulated by two to three imidazoles (1.93 A) and 0.5 S/Cl (2.25 A) per Cu(I). The spectra were insensitive to the presence of added bromide or to exclusion of chloride during preparation. The results establish that the heavy atom scatterer is derived from a sulfur donor. Some evidence was found for an additional O/N scatterer at 2.6 A in the reduced enzyme. A hypothesis for the structure of the copper sites has been proposed involving inequivalent CuA(His)3(H2O)...CuB-(His)2X(H2O) coordination in the oxidized enzyme, which upon reduction loses coordinated water and coordinates a sulfur probably from a methionine.

Carbonmonoxy dopamine beta-hydroxylase. Structural characterization by Fourier transform infrared, fluorescence, and x-ray absorption spectroscopy.

The carbon monoxide complex of ascorbate-reduced dopamine beta-hydroxylase has been prepared and characterized by Fourier transform infrared, fluorescence, and x-ray absorption spectroscopies. CO has previously been shown to be a competitive inhibitor with respect to O2, and binds to only one of the two copper atoms/active site (Blackburn, N. J., Pettingill, T. M., Seagraves, K. S., and Shigeta, R. T. (1990) J. Biol. Chem. 265, 15383-15386). Thus, it acts as an excellent probe of the O2-binding site. A single C-O infrared absorption band is observed at 2089 cm-1, shifting by 46 cm-1 to lower energy on substitution with either 13C16O or 12C18O. The 13C isotope shift is reversed to the position expected for 12CO upon vacuum flushing with 12CO gas, indicating that formation of the CO adduct is a fully reversible process. Binding of the substrate tyramine does not eliminate the infrared peak but causes a 3-cm-1 shift to lower energy. On the other hand, binding of a bifunctional inhibitor which cross-links the substrate and O2-binding site does eliminate the CO peak. These data, in conjunction with the competitive nature of CO binding with respect to O2, identify the CO-binding site as the O2-binding site, and place it in close proximity to the substrate-binding site. CO-dopamine beta-hydroxylase exhibits no luminescence in the visible region, suggesting a structure different from carbonmonoxy hemocyanin, and in all probability mononuclear. Analysis of extended x-ray absorption spectroscopy data is most consistent with an average coordination per Cu of 2-3 histidines, 0.5 CO, and 0.5 S atoms as ligands, and absorption edge comparisons indicates pseudo-4 coordination as the most likely geometry at each Cu(I) center. The results can be interpreted by a model involving inequivalent 4-coordination at each Cu(I) center in the CO adduct with CuAHis3S...CuBHis2CO-X as the coordination most consistent with all of the data.

Characterization of a carbon monoxide complex of reduced dopamine beta-hydroxylase. Evidence for inequivalence of the Cu(I) centers.

Ascorbate-reduced dopamine beta-hydroxylase (DBH) is inhibited by CO in a competitive manner with respect to molecular O2. Measurement of the stoichiometry of CO binding indicates 0.50 CO bound per Cu(I), which provides the first evidence that the Cu(I) centers in the reduced enzyme are structurally inequivalent. FTIR spectroscopy has been used to detect an infrared absorption band characteristic of coordinated CO, with v(CO) = 2089 cm-1. Comparison of this frequency with those of other Cu(I)-carbonyls in both inorganic and protein systems suggests a coordination site with fewer or less basic ligands than the 3-histidine site of carbon-monoxy hemocyanin.