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The tyrosyl radicals generated in reactions of ethyl hydrogen peroxide with both native and indomethacin-pretreated prostaglandin H synthase 1 (PGHS-1) were examined by low-temperature electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies. In the reaction of peroxide with the native enzyme at 0 degrees C, the tyrosyl radical EPR signal underwent a continuous reduction in line width and lost intensity as the incubation time increased, changing from an initial, 35-G wide doublet to a wide singlet of slightly smaller line width and finally to a 25-G narrow singlet. The 25-G narrow singlet produced by self-inactivation was distinctly broader than the 22-G narrow singlet obtained by indomethacin treatment. Analysis of the narrow singlet EPR spectra of self-inactivated and indomethacin-pretreated enzymes suggests that they reflect conformationally distinct tyrosyl radicals. ENDOR spectroscopy allowed more detailed characterization by providing hyperfine couplings for ring and methylene protons. These results establish that the wide doublet and the 22-G narrow singlet EPR signals arise from tyrosyl radicals with different side-chain conformations. The wide-singlet ENDOR spectrum, however, is best accounted for as a mixture of native wide-doublet and self-inactivated 25-G narrow-singlet species, consistent with an earlier EPR study [DeGray et al. (1992) J. Biol. Chem. 267, 23583-23588]. We conclude that a tyrosyl residue other than the catalytically essential Y385 species is most likely responsible for the indomethacin-inhibited, narrow-singlet spectrum. Thus, this inhibitor may function by redirecting radical formation to a catalytically inactive side chain. Either radical migration or conformational relaxation at Y385 produces the 25-G narrow singlet during self-inactivation. Our ENDOR data also indicate that the catalytically active, wide-doublet species is not hydrogen bonded, which may enhance its reactivity toward the fatty-acid substrate bound nearby.

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 double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies were used to study whether protons in the immediate protein environment around CuA in cytochrome c oxidase are susceptible to solvent exchange. The enzyme was incubated in buffered D2O under resting or turnover conditions for 90 min and then frozen to quench the hydrogen/deuterium-exchange process. ENDOR spectra of the deuterated sample were essentially identical to those of control samples. The ESEEM spectra, however, provided a clear indication of the introduction of deuterium into the CuA environment following incubation in buffered D2O. The extent of deuterium incorporation was not affected by enzyme turnover. An analysis of the ESEEM data indicated that water is in reasonably close proximity to the CuA site, but not in the immediate coordination sphere of the metal(s). We estimate a minimum distance of 5.4 A between the CuA center and the protein/water interface. This relatively short surface separation distance is consistent with the role of CuA as the immediate oxidant of cytochrome c in the cytochrome oxidase (Hill, B. C. (1991) J. Biol. Chem. 266, 2219-2226).

The nuclear modulation effect in pulsed EPR spectroscopy was used to study the type 2 copper binding site in the mercury derivative of laccase (MDL) in which the type 1 copper is substituted by Hg(II). By comparing the three-pulse electron spin-echo modulations and Fourier transform spectra of MDL and several model compounds, we conclude that the imidazole groups of two histidyl amino acid residues are equatorially coordinated to Cu(II) in the type 2 site. Computer simulations of these data suggest that the remote nonbonding nitrogens of the two imidazoles possess nuclear quadrupole parameters e2qQ = 1.47 MHz and eta = 0.83. A(iso) values of these two nitrogens are not identical, being 1.5 and 2.0 MHz. We have also used samples of the enzyme exchanged with D2O to examine the coordination of the water to the type 2 copper site. The deuterium modulation that is resolved by taking the ratio of the time domain ESEEM data from native and D2O-exchanged enzyme indicates that there is an equatorial water ligand, and further data show that this water is displaced by azide.