Identification of the Cu2+ binding sites in the N-terminal domain of the prion protein by EPR and CD spectroscopy.

Recent evidence indicates that the prion protein (PrP) plays a role in copper metabolism in the central nervous system. The N-terminal region of human PrP contains four sequential copies of the highly conserved octarepeat sequence PHGGGWGQ spanning residues 60-91. This region selectively binds divalent copper ions (Cu(2+)) in vivo. To elucidate the specific mode and site of binding, we have studied a series of Cu(2+)-peptide complexes composed of 1-, 2-, and 4-octarepeats and several sub-octarepeat peptides, by electron paramagnetic resonance (EPR, conventional X-band and low-frequency S-band) and circular dichroism (CD) spectroscopy. At pH 7.45, two EPR active binding modes are observed where the dominant mode appears to involve coordination of three nitrogens and one oxygen to the copper ion, while in the minor mode two nitrogens and two oxygens coordinate. ESEEM spectra demonstrate that the histidine imidazole contributes one of these nitrogens. The truncated sequence HGGGW gives EPR and CD that are indistinguishable from the dominant binding mode observed for the multi-octarepeat sequences and may therefore comprise the fundamental Cu(2+) binding unit. Both EPR and CD titration experiments demonstrate rigorously a 1:1 Cu(2+)/octarepeat binding stoichiometry regardless of the number of octarepeats in a given peptide sequence. Detailed spin integration of the EPR signals demonstrates that all of the bound Cu(2+) is detected thereby ruling out strong exchange coupling that is often found when there is imidazolate bridging between paramagnetic metal centers. A model consistent with these data is proposed in which Cu(2+) is bound to the nitrogen of the histidine imidazole side chain and to two nitrogens from sequential glycine backbone amides.

Demonstration of a conserved histidine and two water ligands at the Mn2+ site in Diocleinae lectins by pulsed EPR spectroscopy.

Lectins from the Diocleinae subtribe, including Canavalia brasiliensis, Canavalia bonariensis, Canavalia grandiflora, Cratylia floribunda, Dioclea grandiflora, Dioclea guianensis, Dioclea rostrata, Dioclea violacea, and Dioclea virgata, have been recently isolated and characterized in terms of their carbohydrate binding specificities. Although all of the lectins are Man/Glc specific, they possess different biological activities. In the present study, electron paramagnetic resonance (EPR) spectroscopy demonstrates that all nine Diocleinae lectins contain Mn2+. The spectra of C. floribunda and D. rostrata suggest Mn2+ site symmetry different from that of the other seven lectins. However, electron spin-echo envelope modulation (ESEEM) spectroscopy indicates that all nine lectins are coordinated to a histidyl imidazole, with similar electron-nuclear coupling to the Mn2+-bound imidazole nitrogen. ESEEM also demonstrates ligation of two water molecules to Mn2+ in all nine Diocleinae lectins. Thus, the EPR and ESEEM data indicate the presence of a Mn2+ binding site in the above Diocleinae lectins with a conserved histidine residue and two water ligands.

Electron spin-echo envelope modulation study of multicrystalline Cu(2+)-insulin: effects of Cd(2+) on the nuclear quadrupole interaction of the Cu(2+)-coordinated imidazole remote nitrogen.

A comparison of electron spin-echo envelope modulation (ESEEM) spectra from multi-crystalline Cu(2+)-insulin with and without additional Cd(2+) show a dramatic change in the quadrupole coupling parameters of the remote nitrogens of the two histidine imidazoles that ligate to copper. Without Cd(2+), the quadrupole parameters are like those observed in blue copper proteins and in copper substituted lactoferrin. With Cd(2+) soaked into the Cu(2+)-insulin crystals, the quadrupole parameters are similar to those found in galactose oxidase. Theoretical simulations of ESEEM spectra guided by structure modeling suggest that these changes originate from differences in the hydrogen bonding environments of the imidazole remote nitrogen. In addition, a compilation of results from previous ESEEM studies of copper proteins reveals that the asymmetry parameter, eta, may be an indicator of type of hydrogen bond the imidazole remote nitrogen makes. When eta > or = 0.9, the nitrogen hydrogen bonds to water, whereas when eta < 0.9, the nitrogen hydrogen bonds to the protein.

Identification of the ligands to the ferric heme of Chlamydomonas chloroplast hemoglobin: evidence for ligation of tyrosine-63 (B10) to the heme.

We have studied the unusual heme ligand structure of the ferric forms of a recombinant Chlamydomonas chloroplast hemoglobin and its several single-amino acid mutants by EPR, optical absorbance, and resonance Raman spectroscopy. The helical positions of glutamine-84, tyrosine-63, and lysine-87 are suggested to correspond to E7, B10, and E10, respectively, in the distal heme pocket on the basis of amino acid sequence comparison of mammalian globins. The protein undergoes a transition with a pK of 6.3 from a six-coordinate high-spin aquomet form at acidic pH to a six-coordinate low-spin form. The EPR signal of the low-spin form for the wild-type protein is absent for the Tyr63Leu mutant, suggesting that the B10 tyrosine in the wild-type protein ligates to the heme as tyrosinate. For the Tyr63Leu mutant, a new low-spin signal resembling that of alkaline cytochrome c (a His-heme-Lys species) is resolved, suggesting that the E10 lysine now coordinates to the heme. In the wild-type protein, the oxygen of the tyrosine-63 side chain is likely to share a proton with the side chain of lysine-87, suggested by the observation of a H/D sensitive resonance Raman line at 502 cm(-)(1) that is tentatively assigned as a vibrational mode of the Fe-O bond between the iron and the tyrosinate. We propose that the transition from the high-spin to the low-spin form of the protein occurs by deprotonation and ligation to the heme of the B10 tyrosine oxygen, facilitated by strong interaction with the E10 lysine side chain.

Chlamydomonas chloroplast ferrous hemoglobin. Heme pocket structure and reactions with ligands.

We report the optical and resonance Raman spectral characterization of ferrous recombinant Chlamydomonas LI637 hemoglobin. We show that it is present in three pH-dependent equilibrium forms including a 4-coordinate species at acid pH, a 5-coordinate high spin species at neutral pH, and a 6-coordinate low spin species at alkaline pH. The proximal ligand to the heme is the imidazole group of a histidine. Kinetics of the reactions with ligands were determined by stopped-flow spectroscopy. At alkaline pH, combination with oxygen, nitric oxide, and carbon monoxide displays a kinetic behavior that is interpreted as being rate-limited by conversion of the 6-coordinate form to a reactive 5-coordinate form. At neutral pH, combination rates of the 5-coordinate form with oxygen and carbon monoxide were much faster (>10(7) microM-1 s-1). The dissociation rate constant measured for oxygen is among the slowest known, 0.014 s-1, and is independent of pH. Replacement of the tyrosine 63 (B10) by leucine or of the putative distal glutamine by glycine increases the dissociation rate constant 70- and 30-fold and increases the rate of autoxidation 20- and 90-fold, respectively. These results are consistent with at least two hydrogen bonds stabilizing the bound oxygen molecule, one from tyrosine B10 and the other from the distal glutamine. In addition, the high frequency (232 cm-1) of the iron-histidine bond suggests a structure that lacks any proximal strain thus contributing to high ligand affinity.

The heme environment in barley hemoglobin.

To elucidate the environment and ligand structure of the heme in barley hemoglobin (Hb), resonance Raman and electron paramagnetic resonance spectroscopic studies have been carried out. The heme is shown to have bis-imidazole coordination, and neither of the histidines has imidazolate character. Barley Hb has a unique heme environment as judged from the Fe-CO and C-O stretching frequencies in the CO complex. Two Fe-CO stretching modes are observed with frequencies at 534 and 493 cm-1, with relative intensities that are pH sensitive. The 534 cm-1 conformer shows a deuterium shift, indicating that the iron-bound CO is hydrogen-bonded, presumably to the distal histidine. A C-O stretching mode at 1924 cm-1 is assigned as being associated with the 534 cm-1 conformer. Evidence is presented that the high Fe-CO and low C-O stretching frequencies (534 and 1924 cm-1, respectively) arise from a short hydrogen bond between the distal histidine and the CO. The 493 cm-1 conformer arises from an open conformation of the heme pocket and becomes the dominant population under acidic conditions when the distal histidine moves away from the CO. Strong hydrogen bonding between the bound ligand and the distal histidine in the CO complex of barley Hb implies that a similar structure may occur in the oxy derivative, imparting a high stability to the bound oxygen. This stabilization is confirmed by the dramatic decrease in the oxygen dissociation rate compared with sperm whale myoglobin.

Sequence-specific changes in the metal site of ferric bleomycin induced by the binding of DNA.

The binding of the iron complex of the antineoplastic glycopeptide bleomycin A2 (Fe-BLM) to calf thymus DNA and the self-complementary oligonucleotides d(CGCGCG) and d(ATATAT) has been studied using optical, EPR, and resonance Raman spectroscopies. An increase in the intensity of the bands at 365 and 384 nm is observed in the optical spectrum of Fe(III)-BLM when the drug binds to either oligonucleotide. However, in the presence of phosphate, this increase is observed only with d(CGCGCG) and not with d(ATATAT). In addition, the gmax feature in the EPR spectrum of low spin Fe(III)-BLM is narrowed in a way suggesting a reduction of possible conformers that the drug can achieve when it is bound to d(CGCGCG) or to calf thymus DNA but not when bound to d(ATATAT). When Fe(III)-BLM is bound to d(CGCGCG), changes in the resonance Raman spectrum of the metal drug complex suggest conformational changes in three of the ligands to iron: the beta-hydroxyhistidyl amide, the pyrimidine, and the axial hydroxide. In addition, the Fe-OH band undergoes narrowing, again consistent, with the reduction of conformers of the drug. No such resonance Raman changes are observed upon binding to d(ATATAT). The changes in the pyrimidine modes upon binding d(CGCGCG) to the drug are consistent with a recently proposed model (Wu, W., Vanderwall, D. E., Turner, C. J., Kozarich, J. W., and Stubbe, J. (1996) J. Am. Chem. Soc. 118, 1281-1294) of DNA recognition by activated bleomycin, HOO-Fe(III)-BLM, in which the pyrimidine moiety of the drug is important for the preferential cleavage of 5'-GpPy-3' sequences.

Dioxygen inactivation of pyruvate formate-lyase: EPR evidence for the formation of protein-based sulfinyl and peroxyl radicals.

We here report EPR studies that provide evidence for radical intermediates generated from the glycyl radical of activated pyruvate formate-lyase (PFL) during the process of oxygen-dependent enzyme inactivation, radical quenching, and protein fragmentation. Upon exposure of active PFL to air, a long-lived radical intermediate was generated, which exhibits an EPR spectrum assigned to a sulfinyl radical (RSO*). The EPR spectrum of a sulfinyl radical was also generated from the activated C418A mutant of PFL, indicating that Cys 418 is not the site of sulfinyl radical formation. Exposure of the activated C419A mutant or C418AC419A double mutant to air on the other hand, resulted in a new EPR spectrum that we assign to the alpha-carbon peroxyl radical (ROO*) of the active-site glycine, G734. These findings suggest that C419 is the site of sulfinyl radical formation and that replacement of this cysteine with alanine results in the accumulation of the carbon peroxyl radical. The results also support the proposal that the peroxyl radical and the sulfinyl radical are intermediates in the oxygen-dependent inactivation and cleavage of the protein. Moreover, these observations are consistent with the hypothesis that C419 and G734 are in close proximity in the activated enzyme and may participate in a glycyl/thiyl radical equilibrium. A mechanism that accounts for the formation of the radical intermediates is proposed.

Cloning, expression, and spectroscopic characterization of Cucumis sativus stellacyanin in its nonglycosylated form.

The cDNA encoding the 182 amino acid long precursor stellacyanin from Cucumis sativus was isolated and characterized. The protein precursor consists of four sequence domains: I, a 23 amino acid hydrophobic N-terminal signal peptide with features characteristic of secretory proteins; II, a 109 amino acid copper-binding domain; III, a 26 amino acid hydroxyproline- and serine-rich peptide characteristic of motifs found in the extension family, extracellular structural glycoproteins found in plant cell walls; and IV, a 22 amino acid hydrophobic extension. Maturation of the protein involves posttranslational processing of domains I and IV. The copper-binding domain (domain II), which shares high sequence identity with other stellacyanins, has been expressed without its carbohydrate attachment sites, refolded from the Escherichia coli inclusion bodies, purified, and characterized by electronic absorption, EPR, ESEEM, and RR spectroscopy. Its spectroscopic properties are nearly identical to those of stellacyanin from the Japanese lacquer tree Rhus vernicifera, the most extensively studied and best characterized stellacyanin, indicating that this domain folds correctly, even in the absence of its carbohydrate moiety. The presence of a hydroxyproline- and serine-rich domain III suggests that stellacyanin may have a function other than that of a diffusible electron transfer protein, conceivably participating in redox reactions localized at the plant cell wall, which are known to occur in response to wounding or infection of the plant.

Role of the divalent metal ion in the NAD:malic enzyme reaction: an ESEEM determination of the ground state conformation of malate in the E:Mn:malate complex.

The conformation of L-malate bound at the active site of Ascaris suum malic enzyme has been investigated by electron spin echo envelope modulation spectroscopy. Dipolar interactions between Mn2+ bound to the enzyme active site and deuterium specifically placed at the 2-position, the 3R-position, and the 3S-position of L-malate were observed. The intensities of these interactions are related to the distance between each deuterium and Mn2+. Several models of possible Mn-malate complexes were constructed using molecular graphics techniques, and conformational searches were conducted to identify conformers of malate that meet the distance criteria defined by the spectroscopic measurements. These searches suggest that L-malate binds to the enzyme active site in the trans conformation, which would be expected to be the most stable conformer in solution, not in the gauche conformer, which would be more similar to the conformation required for oxidative decarboxylation of oxalacetate formed from L-malate at the active site of the enzyme.

Simulation of Mn (II) EPR spectra using a full spin-Hamiltonian approach.

A full spin-Hamiltonian approach was used to calculate continuous-wave EPR spectra of Mn(II) complexes, and a comparison was made with spectra previously simulated by perturbation methods. Successful fits were performed using single values of the zero-field splitting parameter, D. The advantage of the new procedure is that it provides better fits where the magnetic field is not very large compared to zero-field splitting terms.

A chemical modification of cytochrome-c lysines leading to changes in heme iron ligation.

Although 13 lysines of horse cytochrome c are invariant, and three more are extremely conserved, the modification of their side-chain epsilon-amino groups by beta-thiopropionylation caused important changes in protein properties for only three of them; lysines 72,73 and 79. Optical spectroscopy, electron and nuclear paramagnetic resonance, electron spin echo envelope modulation, and molecular weight studies, as well as the unique features of their reaction with cytochrome-c oxidase, indicate that in the oxidized state the modification of these lysines resulted in equilibria between two different states of iron ligation: the native state, in which the metal is coordinated by the methionine-80 sulfur, and a new state in which this ligand is displaced by the sulfhydryl groups of the elongated side chains. The reduction potentials of the TP Lys-72 and the TP Lys-79 derivatives were 201 and 196 millivolt, respectively, indicating that the equilibria favored the sulfhydryl ligated state by 1.5 and 1.7 kcal/mol, respectively. In the ferric state, the protein modified at lysine 72 remained stable as a monomer, but that modified at lysine 73 dimerized rapidly through disulfide bond formation, while the TP Lys-79 cytochrome c dimerized with a half-time of approx. 3 h, both recovering the native-like iron ligation. By contrast, in the ferrous state the monomeric state and the native ligation were preserved in all cases, indicating that the affinity of the cytochrome-c ferrous iron for the methionine-80 sulfur is particularly strong. The dimerized derivatives lost most, but not all, of the capability of the native protein for electron transfer from ascorbate-TMPD to cytochrome-c oxidase.

Electron paramagnetic resonance evidence for a cysteine-based radical in pyruvate formate-lyase inactivated with mercaptopyruvate.

Pyruvate formate-lyase (PFL) is a glycyl radical-containing enzyme that catalyzes the reversible, nonoxidative conversion of pyruvate and CoA into acetyl-CoA and formate. The radical is located on the alpha-carbon of glycine 734 and is required for catalysis. Two cysteine residues, C418 and C419, are also essential for catalysis. Mercaptopyruvate, a biologically relevant pyruvate analog, is shown here to be a mechanism-based inactivator of PFL. Upon addition of mercaptopyruvate to active PFL, an EPR spectrum is generated which exhibits components from two sulfur-based radicals. For one of these radicals, a disulfide radical, the hyperfine coupling to a single beta-methylene hydrogen is resolved in features at g = 2.057 and 2.023. The effects of deuterium labeling of the enzyme on the EPR spectrum for this species are consistent with the new radical being on a cysteine residue, probably cysteine 418 or 419. This spectrum is not formed upon addition of the inactivator to mutant enzymes, C418S and C419S, indicating that both active site cysteines are required for formation of the new radicals. The identity of the second species is also ascribed to be a sulfur-based radical on the basis of the EPR feature found at g = 2.01. Our results constitute the first direct evidence of sulfur-based radical formation in an enzyme. A mechanism for formation of the cysteine-based disulfide radical is proposed which requires the participation of the two active site cysteines as well as the glycyl radical.

Electron spin echo envelope modulation study of oxygenated iron-cobalt hybrid hemoglobins reveals molecular features analogous to those of the oxy ferrous protein.

Two oxygenated iron-cobalt hybrid hemoglobins (Hbs), (alpha Co-O2 beta Fe-O2)2 and (alpha Fe-O2 beta Co-O2)2, were studied by electron spin echo envelope modulation (ESEEM) spectroscopy in order to measure (i) electron-nuclear hyperfine and nuclear quadrupole coupling to the N epsilon of the proximal histidyl imidazole and (ii) nuclear hyperfine coupling to exchangeable 2H in the oxyCo subunits. 14N couplings were found to be smaller in the oxyCo alpha subunits than in the oxyCo beta subunits, suggesting a more ionic and shorter Co-O2 bond in the alpha subunits [Lee et al. (1994) Biochemistry 33, 7609], which correlates with the higher O2 affinity found for (alpha Co beta Fe-O2)2 Hb than for (alpha Fe-O2 beta Co)2 Hb [Imai et al. (1980) J. Mol. Biol. 138, 635]. A smaller nuclear quadrupole coupling constant found for the proximal histidyl N epsilon in the oxyCo alpha subunits also suggests an increase in the overlap between the N epsilon sp2 hybrid and the Co dz2 orbital, i.e., a shorter Co-N epsilon bond, than in the oxyCo beta subunits. On the other hand, the relative orientation of the g and 14N epsilon nuclear quadrupole tensors, obtained by spectral simulation, suggests that the Co-O-O bond angle is similar in the two types of oxyCo subunits. An X-ray crystallographic study of oxyFe Hb A [Shaanan, B. (1982) Nature 296, 683] has also reported similar Fe-O-O bond angles in both alpha and beta subunits, but with shorter Fe-N epsilon and Fe-O2 bonds in the alpha subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrogen exchange of the glycyl radical of pyruvate formate-lyase is catalyzed by cysteine 419.

Pyruvate formate-lyase (PFL) catalyzes the reversible conversion of CoA and pyruvate into acetyl-CoA and formate. Active enzyme contains a glycyl radical whose alpha-hydrogen undergoes rapid exchange with solvent (t1/2 approximately 5 min at 0 degree C). We have investigated this exchange using site-directed mutagenesis and mechanism-based inactivation. Mutation of the active-site cysteine 419 into a serine, which renders the enzyme catalytically inactive, abolishes alpha-hydrogen exchange in the radical. This suggests that the exchange process is not an intrinsic property of the glycyl radical but is a consequence of its interaction with cysteine 419. This residue is also demonstrated to be involved in the transfer of the radical to acetylphosphinate, a mechanism-based inactivator of the enzyme. In contrast, mutation of the other essential cysteine 418 to a serine has no effect on the hydrogen exchange or the transfer of the radical to acetylphosphinate. A mechanism for the hydrogen exchange catalyzed by cysteine 419 consistent with a redox role for this residue in the normal catalytic reaction is proposed.

Cu(II) coordination in arthropod and mollusk green half-methemocyanins analyzed by electron spin-echo envelope modulation spectroscopy.

Hemocyanin (Hc) is a dinuclear copper protein that binds oxygen reversibly. The structure of the Cu(II) site in a derivative of hemocyanin known as green half-met (GHM) has been analyzed using the pulsed EPR technique of electron spin-echo envelope modulation (ESEEM) spectroscopy. The derivative, prepared by treating the native protein with nitrite at low pH, contains a mixed-valent binuclear copper center. It was shown through chemical assays and the ligand exchange reaction products identified by EPR spectroscopy to contain a nitrite ligand bound to Cu(II). The ESEEM spectra of green half-methemocyanins from mollusks and arthropods indicated that three imidazole ligands are coordinated to Cu(II). Therefore, a tetragonal N3O ligand structure (O is an oxygen of nitrite) is proposed. For GHM Hc from the mollusks Octopus vulgaris and Rapana thomasiana, the isotropic nitrogen nuclear hyperfine coupling constant, aiso, for the N delta (or remote) nitrogen of two imidazoles was approximately 1.4 MHz, while for the third, aiso congruent to 2.2 MHz. The difference between the two weaker nitrogens and the single, more strongly coupled nitrogen was smaller by 0.2 MHz in the GHM Hcs from the arthropods Carcinus maenas, Homarus americanus and Panulirus interruptus. The nitrogen nuclear quadrupole coupling constants and asymmetry parameters, e2Qq and eta, for the N delta nitrogens in nearly all cases were near 1.4 MHz and 0.8, respectively, although Rapana thomasiana GHM Hc exhibited a reduction in eta that may indicate weaker hydrogen bonding in the active site of this protein. The g and ACu (copper nuclear hyperfine coupling) values for the derivatives, and the finding of three similar nuclear hyperfine coupling constants for the N delta sites of imidazole ligands, when considered with the orientation-specific information obtained using angle-selection methods for simulation of ESEEM spectra, suggest a distorted tetragonal Cu(II) structure in which three imidazoles and a nitrite ligand are bound near the equatorial plane. The finding that the two molluscan GHM Hcs exhibit differences associated with the remote nitrogen of imidazoles bound to Cu(II) may be related to a structural variability in the active sites of these proteins not found in the arthropodan GHM Hcs examined.

Structural characterization of mononuclear Cu(II) and its nitrite complex in the active site of Carcinus maenas hemocyanin.

The preparation of a mononuclear Cu(II) derivative of Carcinus maenas hemocyanin (Cu(II)-Hc) and a nitrite complex of the derivative (Cu(II)-Hc-NO2-) are described. Several techniques have been used in their characterization, including X-ray absorption, continuous wave (cw) EPR, and electron spin-echo envelope modulation (ESEEM) spectroscopies. EXAFS results for Cu(II)-Hc indicate the presence of three ligands at 1.99 +/- 0.01 A and a fourth one at 2.26 +/- 0.01 A from the copper. The same coordination number and very similar bond lengths were obtained for Cu(II)-Hc-NO2-. On the basis of simulations of three-pulse ESEEM spectra, three equivalent imidazole nitrogens coupled to Cu(II) were identified in Cu(II)-Hc. Upon the binding of nitrite, a decrease in the hyperfine interaction for two of the three imidazole nitrogens was observed by ESSEM. Further, the results of a two-pulse ESEEM experiment are consistent with the assignment of the protons of a water ligand to Cu(II), which is displaced when nitrite is added. An analysis of X-ray absorption K-edge spectra suggests a coordination geometry intermediate between square-planar and tetrahedral for the metal centers in Cu(II)-Hc and Cu(II)-Hc-NO2-, in agreement with the g and ACu values determined by cw-EPR. On the basis of these results, an equivalent structure is suggested for Cu(II)-Hc-NO2- and the Cu(II) site in green half-methemocyanin, a partially oxidized binuclear derivative formed in the reaction of the native protein with nitrite.

Identification of metal ligands in Cu(II)-inhibited Chromobacterium violaceum phenylalanine hydroxylase by electron spin echo envelope modulation analysis of histidine to serine mutations.

Phenylalanine hydroxylase from Chromobacterium violaceum (CVPAH) is known to bind an equivalent of divalent copper. The "metal-free" form of the protein is fully active, and Cu(II) is now shown to be an inhibitor of CVPAH rather than an activator of the enzyme [Carr, R. T., & Benkovic, S. J. (1994) Biochemistry 32, 14132-14138]. On the basis of amino acid sequence homology, the metal binding site may be related to those of rat liver PAH and other eukaryotic pterin-dependent hydroxylases, which require Fe(II) for activity. The conserved histidines at that site in CVPAH, histidines 138 and 143, were each mutated to serines. The mutant enzymes H138S and H143S were both catalytically inactive, but still able to bind Cu(II). Binding studies further demonstrated that both mutant enzymes still bind L-phenylalanine. Electron spin echo envelope modulation (ESEEM) studies on each of the mutants showed the presence of only a single copper-coordinating histidine, rather than the two histidine ligands suggested for the wild-type protein. This result supports a model in which Cu(II) is equatorially ligated to only two histidines in the Cu(II)-inhibited protein and allows us to unambiguously assign histidines 138 and 143 as these ligands. That the enzyme is inactive when these histidines are either bound with copper or when replaced with serines suggests that these histidines perform a catalytic function. Possible catalytic roles for these histidines in the hydroxylation mechanism of pterin-dependent monooxygenases are discussed along with potential future applications of the combination of ESEEM with site-directed mutagenesis.

Electron-nuclear coupling to the proximal histidine in oxy cobalt-substituted distal histidine mutants of human myoglobin.

Electron spin echo envelope modulation (ESEEM) spectroscopy was used to investigate electron-nuclear coupling to the N epsilon of the proximal histidine (F8, His93) imidazole in oxyCo(II)-substituted distal histidine (E7, His64) mutants (His-->Leu, His-->Val, His-->Gly, His-->Gln) and recombinant wild-type human myoglobins (Mbs). Nuclear hyperfine and nuclear quadrupole coupling constants decrease in the order: H64L > H64V > or = H64G approximately H64Q > wild-type. The differences in couplings found for the four mutant proteins are correlated with the differences in polarity of the E7 side chain. On the basis of the relative orientation of the nuclear quadrupole and g tensors, obtained by computer simulation of ESEEM spectra, the Co-O-O bond angle of H64G and H64Q appears to be similar to that of oxyCo sperm whale Mb (and possibly wild-type human Mb) at room temperature [Hori et al. (1982) J. Biol. Chem. 257, 3636], while that in H64V and H64L is more obtuse. ESEEM measurements in D2O demonstrate the presence of a hydrogen bond between the distal histidine and bound O2 in the wild-type protein, as was found in oxyCo sperm whale and horse Mbs [Lee et al. (1992) Biochemistry 31, 7274]. This hydrogen bond leads to a reduction in the N epsilon coupling in the wild-type protein as compared to that in the E7 mutants. No hyperfine-coupled deuterons were found in any of the mutants, and therefore, the proposed hydrogen bond between bound O2 and the distal glutamine in H64Q [Ikeda-Saito et al. (1991) J. Biol. Chem. 266, 23641] could not be substantiated.