Model for the Porphyrin-DNA Binding Site: ENDOR Investigations of Cu-Porphyrins Binding to DNA

Abstract

Proton ENDOR has been observed from frozen solutions (ca. 38K°) of copper meso-(4-N-tetra-methylpyridyl)porphyrin (CuTMpyP(4)) complexed with Salmon sperm DNA in water and D₂O. Lines from exchangeable protons of the DNA bases have been observed in these ENDOR spectra. Analyses of these ENDOR data show that the separations of these DNA protons from the copper atom are between 3.76 and 3.84 A with angles of 19.5 to 22.5 degrees between the Cu-H vectors and the g_z axis. A distant ENDOR response has also been observed from phosphorous nuclei in the DNA backbone. We estimate that the phosphorous atoms producing this ENDOR signal are 7.5–10 Å from the copper center of the porphyrin. These ENDOR data combined with results from an earlier NMR investigation (1) have been used to construct a computer simulated model of the binding site in which the porphyrin is partially intercalated and extends into the major groove of DNA. The two GC base pairs at this site are slightly inequivalent. For each, the G imino proton and one of the C amino protons are at appropriate positions to account for the ENDOR signals arising from exchangeable protons. It is unlikely that this inequivalence would persist at room temperature where dynamic processes would give an apparently symmetric interaction. Although the model accounts for all reported experimental data involving tetracationic porphyrin species which have been suggested to be intercalators, it is not a unique solution.     

5'-Deoxyadenosine contacts the substrate radical intermediate in the active site of ethanolamine ammonia-lyase: 2H and 13C electron nuclear double resonance studies

The mechanism of propagation of the radical center between the cofactor, substrate, and product in the adenosylcobalamin- (AdoCbl) dependent reaction of ethanolamine ammonia-lyase has been probed by pulsed electron nuclear double resonance (ENDOR) spectroscopy. The radical of S-2-aminopropanol, which appears in the steady state of the reaction, was used in ENDOR experiments to determine the nuclear spin transition frequencies of (2)H introduced from either deuterated substrate or deuterated coenzyme and of (13)C introduced into the ribosyl moiety of AdoCbl. A (2)H doublet (1.4 MHz splitting) was observed centered about the Larmor frequency of (2)H. Identical ENDOR frequencies were observed for (2)H irrespective of its mode of introduction into the complex. A (13)C doublet ENDOR signal was observed from samples prepared with [U-(13)C-ribosyl]-AdoCbl. The (13)C coupling tensor obtained from the ENDOR powder pattern shows that the (13)C has scalar as well as dipole-dipole coupling to the unpaired electron located at C1 of S-2-aminopropanol. The dipole-dipole coupling is consistent with a distance of 3.4+/-0.2 A between C1 of the radical and C5' of the labeled cofactor component. These results establish that the C5' carbon of the 5'-deoxyadenosyl radical moves approximately 7 A from its position as part of AdoCbl to a position where it is in contact with C1 of the substrate which lies approximately 12 A from the Co(2+) of cob(II)alamin. These findings are also consistent with the contention that 5'-deoxyadenosine is the sole mediator of hydrogen transfers in ethanolamine ammonia-lyase.     

Radicals associated with the catalytic intermediates of bovine cytochrome c oxidase

Two radicals have been detected previously by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies in bovine cytochrome oxidase after reaction with hydrogen peroxide, but no correlation could be made with predicted levels of optically detectable intermediates (P_M, F and F) that are formed. This work has been extended by optical quantitation of intermediates in the EPR/ENDOR sample tubes, and by comparison with an analysis of intermediates formed by reaction with carbon monoxide in the presence of oxygen. The narrow radical, attributed previously to a porphyrin cation, is detectable at low levels even in untreated oxidase and increases with hydrogen peroxide treatments generally. It is presumed to arise from a side-reaction unrelated to the catalytic intermediates. The broad radical, attributed previously to a tryptophan radical, is observed only in samples with a significant level of F but when F is generated with hydrogen peroxide, is always accompanied by the narrow radical. When P_M is produced at high pH with CO/O₂, no EPR-detectable radicals are formed. Conversion of the CO/O₂-generated P_M into F when pH is lowered is accompanied by the appearance of a broad radical whose ENDOR spectrum corresponds to a tryptophan cation. Quantitation of its EPR intensity indicates that it is around 3% of the level of F determined optically. It is concluded that low pH causes a change of protonation pattern in P_M which induces partial electron redistribution and tryptophan cation radical formation in F. These protonation changes may mimic a key step of the proton translocation process.     

Radicals associated with the catalytic intermediates of bovine cytochrome c oxidase

Two radicals have been detected previously by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies in bovine cytochrome oxidase after reaction with hydrogen peroxide, but no correlation could be made with predicted levels of optically detectable intermediates (P(M), F and F(z.rad;)) that are formed. This work has been extended by optical quantitation of intermediates in the EPR/ENDOR sample tubes, and by comparison with an analysis of intermediates formed by reaction with carbon monoxide in the presence of oxygen. The narrow radical, attributed previously to a porphyrin cation, is detectable at low levels even in untreated oxidase and increases with hydrogen peroxide treatments generally. It is presumed to arise from a side-reaction unrelated to the catalytic intermediates. The broad radical, attributed previously to a tryptophan radical, is observed only in samples with a significant level of F(z.rad;) but when F(z.rad;) is generated with hydrogen peroxide, is always accompanied by the narrow radical. When P(M) is produced at high pH with CO/O(2), no EPR-detectable radicals are formed. Conversion of the CO/O(2)-generated P(M) into F(z.rad;) when pH is lowered is accompanied by the appearance of a broad radical whose ENDOR spectrum corresponds to a tryptophan cation. Quantitation of its EPR intensity indicates that it is around 3% of the level of F(z.rad;) determined optically. It is concluded that low pH causes a change of protonation pattern in P(M) which induces partial electron redistribution and tryptophan cation radical formation in F(z.rad;). These protonation changes may mimic a key step of the proton translocation process.     

Reaction of bovine cytochrome c oxidase with hydrogen peroxide produces a tryptophan cation radical and a porphyrin cation radical

Oxidized bovine cytochrome c oxidase reacts with hydrogen peroxide to generate two electron paramagnetic resonance (EPR) free radical signals (Fabian, M., and Palmer, G. (1995) Biochemistry 34, 13802-13810). These radicals are associated with the binuclear center and give rise to two overlapped EPR signals, one signal being narrower in line width (DeltaHptp = 12 G) than the other (DeltaHptp = 45 G). We have used electron nuclear double resonance (ENDOR) spectrometry to identify the two different chemical species giving rise to these two EPR signals. Comparison of the ENDOR spectrum associated with the narrow signal with that of compound I of horseradish peroxidase (formed by reaction of that enzyme with hydrogen peroxide) demonstrates that the two species are virtually identical. The chemical species giving rise to the narrow signal is therefore identified as an exchange-coupled porphyrin cation radical similar to that formed in horseradish peroxidase compound I. Comparison of the ENDOR spectrum of compound ES (formed by the reaction of hydrogen peroxide with cytochrome c peroxidase) with that of the broad signal indicates that the chemical species giving rise to the broad EPR signal in cytochrome c oxidase is probably an exchange coupled tryptophan cation radical. This is substantiated using H(2)O/D(2)O solvent exchange experiments where the ENDOR difference spectrum of the broad EPR signal of cytochrome c oxidase shows a feature consistent with hyperfine coupling to the exchangeable N(1) proton of a tryptophan cation radical.     

Structure and interactions of amino acid radicals in class I ribonucleotide reductase studied by ENDOR and high-field EPR spectroscopy

This short review compiles high-field electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) studies on different intermediate amino acid radicals, which emerge in wild-type and mutant class I ribonucleotide reductase (RNR) both in the reaction of protein subunit R2 with molecular oxygen, which generates the essential tyrosyl radical, and in the catalytic reaction, which involves a radical transfer between subunits R2 and R1. Recent examples are presented, how different amino acid radicals (tyrosyl, tryptophan, and different cysteine-based radicals) were identified, assigned to a specific residue, and their interactions, in particular hydrogen bonding, were investigated using high-field EPR and ENDOR spectroscopy. Thereby, unexpected diiron-radical centers, which emerge in mutants of R2 with changed iron coordination, and an important catalytic cysteine-based intermediate in the substrate turnover reaction in R1 were identified and characterized. Experiments on the essential tyrosyl radical in R2 single crystals revealed the so far unknown conformational changes induced by formation of the radical. Interesting structural differences between the tyrosyl radicals of class Ia and Ib enzymes were revealed. Recently accurate distances between the tyrosyl radicals in the protein dimer R2 could be determined using pulsed electron-electron double resonance (PELDOR), providing a new tool for docking studies of protein subunits. These studies show that high-field EPR and ENDOR are important tools for the identification and investigation of radical intermediates, which contributed significantly to the current understanding of the reaction mechanism of class I RNR.     

Structure and interactions of amino acid radicals in class I ribonucleotide reductase studied by ENDOR and high-field EPR spectroscopy

This short review compiles high-field electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) studies on different intermediate amino acid radicals, which emerge in wild-type and mutant class I ribonucleotide reductase (RNR) both in the reaction of protein subunit R2 with molecular oxygen, which generates the essential tyrosyl radical, and in the catalytic reaction, which involves a radical transfer between subunits R2 and R1. Recent examples are presented, how different amino acid radicals (tyrosyl, tryptophan, and different cysteine-based radicals) were identified, assigned to a specific residue, and their interactions, in particular hydrogen bonding, were investigated using high-field EPR and ENDOR spectroscopy. Thereby, unexpected diiron-radical centers, which emerge in mutants of R2 with changed iron coordination, and an important catalytic cysteine-based intermediate in the substrate turnover reaction in R1 were identified and characterized. Experiments on the essential tyrosyl radical in R2 single crystals revealed the so far unknown conformational changes induced by formation of the radical. Interesting structural differences between the tyrosyl radicals of class Ia and Ib enzymes were revealed. Recently accurate distances between the tyrosyl radicals in the protein dimer R2 could be determined using pulsed electron-electron double resonance (PELDOR), providing a new tool for docking studies of protein subunits. These studies show that high-field EPR and ENDOR are important tools for the identification and investigation of radical intermediates, which contributed significantly to the current understanding of the reaction mechanism of class I RNR.     

The differences in microenvironments and functions of tyrosine radicals YZ and YD in photosystem II studied by EPR

Electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) were performed to investigate the difference in microenvironments and functions between tyrosine Z (Y(Z)) and tyrosine D (Y(D)). Mn-depletion or Ca(2+)-depletion causes extension of the lifetime of tyrosine radical Y(Z)(*), which can be trapped by rapid freezing after illumination at about 250 K. Above pH 6.5, Y(Z)(*) radical in Mn-depleted PS II shows similar EPR and ENDOR spectra similar to that of Y(D)(*) radical, which are ascribed to a typical neutral tyrosine radical. Below pH 6.5, Y(Z)(*) radical shows quite different EPR and ENDOR spectra. ENDOR spectra show the spin density distribution of the low-pH form of Y(Z)(*) that has been quite different from the high-pH form of Y(Z)(*). The spin density distribution of the low-pH Y(Z)(*) can be explained by a cation radical or the neutral radical induced by strong electrostatic interaction. The pH dependence of the activation energy of the recombination rate between Y(Z)(*) and Q(A)(-) shows a gap of 4.4 kJ/mol at pH 6.0-6.5. In the Ca(2+)-depleted PS II, Y(Z)(*) signal was the mixture of the cation-like and normal neutral radicals, and the pH dependence of Y(Z)(*) spectrum in Ca(2+)-depleted PS II is considerably different from the neutral radical found in Mn-depleted PS II. Based on the recent structure data of cyanobacterial PS II, the pH dependence of Y(Z)(*) could be ascribed to the modification of the local structure and hydrogen-bonding network induced by the dissociation of ASP170 near Y(Z).     

pH-dependent characteristics of Y(Z) radical in Ca(2+)-depleted photosystem II studied by CW-EPR and pulsed ENDOR

The Y(Z)-tyrosine radical was trapped by freezing immediately after illumination in Ca(2+)-depleted Photosystem II (PS II) membranes and the pH-dependent characteristics of the radical were investigated using CW-EPR and pulsed ENDOR. The spectrum of the Y*(Z) radical trapped in the Y*(Z)S(1) state at pH 5.5 was cation-like as reported in Mn-depleted PS II (H. Mino et al., Spectrochim. Acta A 53 (1997) 1465-1483). By illuminating the PS II-retaining S(2) state, the Y*(Z) radical and a broad doublet signal formed in the g approximately 2 region were trapped concomitantly. The spectrum of the trapped Y*(Z) radical in the Y*(Z)S(2) state was cation-like at pH 5.5 but the pulsed ENDOR measurements reveals the involvement of the neutral Y*(Z) radical in the doublet signal. At pH 7.0, the resulting Y*(Z) signal was the mixture of the cation-like and neutral radical spectra, and considerably different from the neutral radical found in Mn-depleted PS II. pH-Dependent changes in the properties of the Y*(Z) radical are discussed in relation to the redox events occurring in Ca(2+)-depleted PS II.     

Free radical formation in single crystals of 2'-deoxyguanosine 5'-monophosphate tetrahydrate disodium salt: an EPR/ENDOR study.

Single crystals of 2'-deoxyguanosine 5'-monophosphate were X-irradiated at 10 K and at 65 K, receiving doses between 4.5 and 200 kGy, and studied using K-band EPR, ENDOR, and field-swept ENDOR (FSE) spectroscopy. Evidence for five base-centered and more than nine sugar-centered radicals was found at 10 K following high radiation doses. The base-centered radicals were the charged anion, the N10-deprotonated cation, the C8 H-addition radical, a C5 H-addition radical, and finally a stable radical so far unidentified but with parameters similar to those expected for the charged cation. The sugar-centered radicals were the H-abstraction radicals centered at C1', C2', C3', and C5', an alkoxy radical centered at O3', a C5'-centered radical in which the C5'-O5' phosphoester bond appears to be ruptured, a radical tentatively assigned to a C4'-centered radical involving a sugar-ring opening, as well as several additional unidentified sugar radicals. Most radicals were formed regardless of radiation doses. All radicals formed following low doses (4.5-9 kGy) were also observed subsequent to high doses (100-200 kGy). The relative amount of some of the radicals was dose dependent, with base radicals dominating at low doses, and a larger relative yield of sugar radicals at high doses. Above 200 K a transformation from a sugar radical into a base radical occurred. Few other radical transformations were observed. In the discussion of primary radicals fromed in DNA, the presence of sugar-centered radicals has been dismissed since they are not apparent in the EPR spectra. The present data illustrate how radicals barely traceable in the EPR spectra may be identified due to strong ENDOR resonances. Also, the observation of a stable radical with parameters similar to those expected for the charge guanine cation is interesting with regard to the nature of the primary radicals stabilized in X-irradiated DNA.     

Photoaccumulation of the PsaB phyllosemiquinone in Photosystem I of Chlamydomonas reinhardtii

Photoaccumulation of membrane preparations of Chlamydomonas reinhardtii at pH 8 and 220 K reduces the primary and secondary electron acceptors in the Photosystem I (PSI) reaction centre, and produces a maximum of two spins per P700⁺. Proton electron nuclear double resonance (ENDOR) spectra demonstrate that the phyllosemiquinone produced is that attributed to the PsaA branch of electron transfer. Photoaccumulation at pH 10 and 220 K produces a maximum of four spins per P700⁺, and proton ENDOR spectra indicate that a second phyllosemiquinone is being photoaccumulated, with markedly different proton hyperfine couplings (hfcs). This phyllosemiquinone is unaffected by mutation of PsaAW693, confirming that it does not arise from the PsaA branch of electron transfer, and we therefore attribute it to the PsaB phyllosemiquinone.     

Study of catalytic site structure and diffusion of radicals in porous heterogenous systems with ESR,ENDOR and ESE

The generation of paramagnetic products by adsorption of quinones on activated catalysts has been used for the diagnostic of Lewis acid sites. It has been shown that the application of ENDOR, ESE, and 2 mm-band ESR are extremely effective methods for studying the nature of observed radical species and their environment. Two applications of the ESE method for studying the diffusion of spin probes in porous media are considered. The measurement of effective diffusion coefficients of radical probes in specimens of various heterogeneous systems is described. It has been found that effective diffusion coefficients depend strongly on the mean value of silica gel pore sizes and the mobility of the probe inside the pore.     

Chlorophyll and carotenoid radicals in photosystem II studied by pulsed ENDOR

The stable carotenoid cation radical (Car(*+)) and chlorophyll cation radical (Chl(Z)(*+)) in photosystem II (PS II) have been studied by pulsed electron nuclear double resonance (ENDOR) spectroscopy. The spectra were essentially the same for oxygen-evolving PS II and Mn-depleted PS II. The radicals were generated by illumination given at low temperatures, and the ENDOR spectra were attributed to Car(*)(+) and Chl(Z)(*+) on the basis of their characteristic behavior with temperature as demonstrated earlier [Hanley et al. (1999) Biochemistry 38, 8189-8195]: i.e., (a) the Car(*)(+) alone was generated by illumination at < or =20 K, while Chl(Z)(*+) alone was generated at 200 K, and (b) warming of the sample containing the Car(*+) to 200 K resulted in the loss of the signal attributable to Car(*+) and its replacement by a spectrum attributable to the Chl(Z)(*+). A map of the hyperfine structure of Car(*+) in PS II and in organic solvent was obtained. The largest observed hyperfine splitting for Car(*+) in either environment was in the order of 8-9 MHz. Thus, the spin density on the cation is proposed to be delocalized over the carotenoid molecule. The pulsed ENDOR spectrum of Chl(Z)(*)(+) was compared to that obtained from a Chl a cation in frozen organic solvent. The hyperfine coupling constants attributed to the beta-protons at position 17 and 18 are well resolved from Chl(Z)(*+) in PS II (10. 8 and 14.9 MHz) but not in Chl a(*+) in organic solvent (12.5 MHz). This suggests a more defined conformation of ring IV with respect to the rest of the tetrapyrrole ring plane of Chl(Z)(*+) than Chl a(*+) probably induced by the protein matrix.     

EPR and 57Fe ENDOR investigation of 2Fe ferredoxins from Aquifex aeolicus

We have employed EPR and a set of recently developed electron nuclear double resonance (ENDOR) spectroscopies to characterize a suite of [2Fe?C2S] ferredoxin clusters from Aquifex aeolicus (Aae Fd1, Fd4, and Fd5). Antiferromagnetic coupling between the Fe^II, S?=?2, and Fe^III, S?=?5/2, sites of the [2Fe?C2S]⁺ cluster in these proteins creates an S?=?1/2 ground state. A complete discussion of the spin-Hamiltonian contributions to g includes new symmetry arguments along with references to related FeS model compounds and their symmetry and EPR properties. Complete ⁵⁷Fe hyperfine coupling (hfc) tensors for each iron, with respective orientations relative to g, have been determined by the use of ??stochastic?? continuous wave and/or ??random hopped?? pulsed ENDOR, with the relative utility of the two approaches being emphasized. The reported hyperfine tensors include absolute signs determined by a modified pulsed ENDOR saturation and recovery (PESTRE) technique, RD-PESTRE??a post-processing protocol of the ??raw data?? that comprises an ENDOR spectrum. The ⁵⁷Fe hyperfine tensor components found by ENDOR are nicely consistent with those previously found by M?ssbauer spectroscopy, while accurate tensor orientations are unique to the ENDOR approach. These measurements demonstrate the capabilities of the newly developed methods. The high-precision hfc tensors serve as a benchmark for this class of FeS proteins, while the variation in the ⁵⁷Fe hfc tensors as a function of symmetry in these small FeS clusters provides a reference for higher-nuclearity FeS clusters, such as those found in nitrogenase.     

Electron paramagnetic resonance and electron nuclear double resonance spectroscopic identification and characterization of the tyrosyl radicals in prostaglandin H synthase 1

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.     

On the electronic structure of the primary electron donor in bacterial photosynthesis – the bacteriochlorophyll dimer as viewed by EPR/ENDOR methods

The primary electron donor (D) plays a prominent role in electron transfer reactions in the primary processes of photosynthesis. In purple photosynthetic bacteria D is a dimer of bacteriochlorophyll molecules. Investigations on its electronic structure using EPR and ENDOR (electron nuclear double resonance) methods are summarized, focussing on results obtained in the last six years. These encompass studies on the cation radical (D+·) of mutants in which the immediate environment of D is modified through mutagenesis, particularly hydrogen bond and heterodimer mutants. Models using these results to describe the electronic interaction of the dimer halves are discussed. Also, High-Field (95 GHz) EPR to obtain the G-tensor of D+· is addressed. Furthermore, ENDOR on the photoexcited triplet state of D (DT), which in some aspects could serve as a model for the excited singlet state, are discussed. Different approaches towards correlating the electronic structure with function, in particular with the rates of electron transfer reactions, are described.     

Carotenoid radical cations and dications: EPR, optical, and electrochemical studies

Investigations of the structure and properties of paramagnetic carotenoid radical cations and diamagnetic carotenoid dications using electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) spectroscopy in conjunction with electrochemical, optical, and HPLC measurements, and molecular orbital calculations are described. These methods were applied to determine how the carotenoid radical cations and dications can be formed, their electron-transfer properties and stability in various media, and the mechanism by which carotenoid radical cations can isomerize.     

ENDOR spectroscopic studies of stable semiquinone radicals bound to the Escherichia coli cytochrome bo3 quinol oxidase.

The putative oxidation of ubiquinol by the cytochrome bo3 terminal oxidase of Escherichia coli in sequential one-electron steps requires stabilization of the semiquinone. ENDOR spectroscopy has recently been used to study the native ubisemiquinone radical formed in the cytochrome bo3 quinone-binding site [Veselov, A.V., Osborne, J.P., Gennis, R.B. & Scholes, C.P. (2000) Biochemistry 39, 3169-3175]. Comparison of these spectra with those from the decyl-ubisemiquinone radical in vitro indicated that the protein induced large changes in the electronic structure of the ubisemiquinone radical. We have used quinone-substitution experiments to obtain ENDOR spectra of ubisemiquinone, phyllosemiquinone and plastosemiquinone anion radicals bound at the cytochrome bo3 quinone-binding site. Large changes in the electronic structures of these semiquinone anion radicals are induced on binding to the cytochrome bo3 oxidase. The changes in electronic structure are, however, independent of the electronic structures of these semiquinones in vitro. Thus it is shown to be the structure of this binding site in the protein, not the covalent structure of the bound quinone, that determines the electronic structure of the protein-bound semiquinone.     

Electron-nuclear double resonance in melanins.

Electron-nuclear double resonance (ENDOR) signals from matrix protons interacting with the stable free radicals of "A"- and "B"-type melanins have been observed as a function of pH. In all samples the single line is similar in width and unusually narrow. The ENDOR reduction varies by more than a factor of 10, indicating a large sensitivity of relaxation properties of melanin to sample type. Signals were observed over a wide range of experimental conditions with good signal-to-noise ratio, establishing feasibility for further more detailed ENDOR studies. Incubation in D2O resulted in little change, indicating that the free radical is well buried or protected. No resolved hyperfine structure was seen, consistent with the generally accepted view that melanin is a heterogeneous polymer.