The interaction of Trolox C, a water-soluble vitamin E analog, with ferrylmyoglobin: reduction of the oxoferryl moiety.

The oxidation of the heme iron of metmyoglobin by H2O2 yields an oxo ferryl complex (FeIV = O), similar to Compound II of peroxidases, as well as a protein radical; this high oxidation state of myoglobin is known as ferrylmyoglobin. The interaction of Trolox, a water-soluble vitamin E analog, with ferrylmyoglobin entailed two sequential one-electron oxidations of the phenolic antioxidant with intermediate formation of a phenoxyl radical and accumulation of a quinone end product. These oxidation reactions were linked to individual reductions of ferrylmyoglobin to metmyoglobin, as indicated by the value of the relationship [metmyoglobin]formed/[Trolox]consumed: 1.92 +/- 0.28. The Trolox-mediated reduction of ferrylmyoglobin to metmyoglobin could proceed directly, i.e., electron transfer from the phenolic-OH group in Trolox to the oxoferryl moiety, or indirectly, i.e., sequential electron transfer from Trolox to a protein radical to the oxoferryl moiety. The former mechanism is supported by the finding that the high oxidation heme iron is reduced under conditions where the tyrosyl residues are blocked by o-acetylation and when hemin is substituted for myoglobin. The latter mechanism is consistent with the following observations: (a) the EPR signal ascribed to the protein radical is suppressed by Trolox, with the concomitant appearance of the EPR spectrum of the Trolox phenoxyl radical and (b) the rate of ferrylmyoglobin reduction by Trolox is decreased with increasing number of tyrosyl residues in the proteins of horse myoglobin (titrated by o-acetylation) and sperm whale myoglobin. The apparent discrepancy between these observations can be reconciled by considering that both electrophilic centers in ferrylmyoglobin--the oxoferryl heme moiety and the protein radical--function independently of each other and that recovery of ferrylmyoglobin by Trolox could be effected through the tyrosyl residues, albeit at slower rates. The mechanistic aspects of these results are discussed in terms of the two main redox transitions in the myoglobin molecule encompassing valence changes of the heme iron and electron transfer of the tyrosyl residue in the protein and linked to the two sequential one-electron oxidations of Trolox.     

Glutathione-dependent reduction of peroxides during ferryl- and met-myoglobin interconversion: a potential protective mechanism in muscle

Met-myoglobin is oxidized both by H2O2 and other hydroperoxides to a species with a higher iron valency state and the spectral characteristics of ferryl-myoglobin. Glutathione (GSH) reduces the latter species back to met-myoglobin with parallel oxidation to its disulfide (GSSG) but cannot reduce met-myoglobin to ferrous myoglobin. Under aerobic conditions, the GSH-mediated reduction of ferry-myoglobin is associated with O2 consumption and amounts of GSSG are formed far in excess over that of the peroxide added. Under anaerobic conditions, this ratio is close to unity. These results are interpreted in terms of a one-electron redox process involving the reduction of ferryl-myoglobin to met-myoglobin and the one-electron oxidation of GSH to its thiyl radical. Further reactions of thiyl radicals are influenced by the presence of oxygen which will be the determining factor in the ratio H2O2 added/GSSG formed. It is suggested that, when oxygen is limiting, myoglobin may serve as a protector of muscle cells against peroxides and other oxidants.     

Oxidation of myosin by haem proteins generates myosin radicals and protein cross-links

Previous studies have reported that myosin can be modified by oxidative stress and particularly by activated haem proteins. These reactions have been implicated in changes in the properties of this protein in food samples (changes in meat tenderness and palatability), in human physiology (alteration of myocyte function and force generation) and in disease (e.g. cardiomyopathy, chronic heart failure). The oxidant species, mechanisms of reaction and consequences of these reactions are incompletely characterized. In the present study, the nature of the transient species generated on myosin as a result of the reaction with activated haem proteins (horseradish peroxidase/H2O2) and met-myoglobin/H2O2) has been investigated by EPR spectroscopy and amino-acid consumption, product formation has been characterized by HPLC, and changes in protein integrity have been determined by SDS/PAGE. Multiple radical species have been detected by EPR in both the presence and the absence of spin traps. Evidence has been obtained for the presence of thiyl, tyrosyl and other unidentified radical species on myosin as a result of damage-transfer from oxidized myoglobin or horseradish peroxidase. The generation of thiyl and tyrosyl radicals is consistent with the observed consumption of cysteine and tyrosine residues, the detection of di-tyrosine by HPLC and the detection of both reducible (disulfide bond) and non-reducible cross-links between myosin molecules by SDS/PAGE. The time course of radical formation on myosin, product generation and cross-link induction are consistent with these processes being interlinked. These changes are consistent with the altered function and properties of myosin in muscle tissue exposed to oxidative stress arising from disease or from food processing.     

Sulfinyl radical formation from the reaction of cysteine and glutathione thiyl radicals with molecular oxygen

Using Electron Spin Resonance spectroscopy at low temperatures, we find that thiyl radicals resulting from irradiation of frozen aqueous solutions of a variety of thiols, including cysteine, glutathione, and penicillamine react with oxygen to form sulfinyl (RSO.) radicals. The identity of the cysteine sulfinyl radical has been confirmed by the use of molecular oxygen isotopically labeled with 17O. Previous workers have suggested the reaction of thiyl radicals and molecular oxygen resulted in the formation of the potentially damaging thiol peroxyl radical, RSOO.; our work shows no evidence for this species. The sulfinyl radicals are suggested to result from a direct reaction between thiyl radicals and molecular oxygen. This reaction results in the cleavage of the dioxygen bond.     

An ESR investigation of the reactions of glutathione, cysteine and penicillamine thiyl radicals: competitive formation of RSO., R., RSSR-., and RSS(.)

The reactions of the cysteine, glutathione and penicillamine thiyl radicals with oxygen and their parent thiols in frozen aqueous solutions have been elucidated through electron spin resonance spectroscopy. The major sulfur radicals observed are: (1) thiyl radicals, RS.; (2) disulfide radical anions. RSSR-.; (3) perthiyl radicals, RSS. and upon introduction of oxygen; (4) sulfinyl radicals, RSO., where R represents the remainder of the cysteine, glutathione or penicillamine moiety. The radical product observed depends on the pH, concentration of thiol, and presence or absence of molecular oxygen. We find that the sulfinyl radical is a ubiquitous intermediate in the free radical chemistry of these important biological compounds, and also show that peroxyl radical attack on thiols may lead to sulfinyl radicals. We elaborate the observed reaction sequences that lead to sulfinyl radicals, and, using 17O isotopic substitution studies, demonstrate that the oxygen atom in sulfinyl radicals originates from dissolved molecular oxygen. In addition, the glutathione thiyl radical is found to abstract hydrogen from the alpha-carbon position on the cysteine residue of glutathione to form a carbon-centered radical.     

The interaction of ferritin and myoglobin as inductors of lipid peroxidation. The role of reactive oxygen and nitrogen species

The effect of iron dinitrosyl complexes, S-nitrosoglutathione, and glutathione on free radical oxidation of rat heart mitochondria induced by tert-butyl hydroperoxide and metmyoglobin or their combination with ferritin was studied. It was shown that iron dinitrosyl complexes or the combination of S-nitrosoglutathione and glutathione inhibited most effectively the peroxidation of mitochondrial membranes. It was found that ferritin stimulated the prooxidant action of metmyoglobin. Using EPR spectroscopy, it was established that, in conditions of O2*- generation, the destruction of iron dinitrosyl complexes took place. Iron dinitrosyl complexes also inhibited the formation of thiyl radicals, which appeared during O2*- generation in the system containing glutathione and S-nitrosoglutathione. It is essential that the formation of iron dinitrosyl complexes in this reaction system took place with the involvement of ferritin. It was proposed that the prooxidant action of ferritin and myoglobin could be inverted to the antioxidant one.     

A cysteine residue near the propionate side chain of heme is the radical site in ascorbate peroxidase

The radical scavenger 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO(*)) and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were used in conjunction with mass spectrometry to identify the protein-based radical sites of the H(2)O(2)-tolerant ascorbate peroxidase (APX) of the red alga Galdieria partita and the H(2)O(2)-sensitive stromal APX of tobacco. A cysteine residue in the vicinity of the propionate side chain of heme in both enzymes was labeled with TEMPO(*) and DMPO in an H(2)O(2)-dependent manner, indicating that these cysteine residues form thiyl radicals through interaction of APX with H(2)O(2). TEMPO(*) bound to the cysteine thiyl radicals, and sulfinylated and sulfonylated them. Other oxidized cysteine residues were found in both APXs. Experiments with a cysteine-to-serine point mutation showed that formation of TEMPO adducts and subsequent oxidation of the cysteine residue located near the propionate group of heme leads to loss of enzyme activity, in particular in the Galdieria APX. When treated with glutathione and H(2)O(2), both cysteine residues in both enzymes were glutathionylated. These results suggest that, under oxidative stress in vivo, cysteine oxidation is involved in the inactivation of APXs in addition to the proposed H(2)O(2)-mediated crosslinking of heme to the distal tryptophan residue [Kitajima S, Shimaoka T, Kurioka M & Yokota A (2007) FEBS J274, 3013-3020], and that glutathione protects APX from irreversible oxidation of the cysteine thiol and loss of enzyme activity by binding to the cysteine thiol group.     

Probing the free radicals formed in the metmyoglobin-hydrogen peroxide reaction

The reaction between metmyoglobin and hydrogen peroxide results in the two-electron reduction of H2O2 by the protein, with concomitant formation of a ferryl-oxo heme and a protein-centered free radical. Sperm whale metmyoglobin, which contains three tyrosine residues (Tyr-103, Tyr-146, and Tyr-151) and two tryptophan residues (Trp-7 and Trp-14), forms a tryptophanyl radical at residue 14 that reacts with O2 to form a peroxyl radical and also forms distinct tyrosyl radicals at Tyr-103 and Tyr-151. Horse metmyoglobin, which lacks Tyr-151 of the sperm whale protein, forms an oxygen-reactive tryptophanyl radical and also a phenoxyl radical at Tyr-103. Human metmyoglobin, in addition to the tyrosine and tryptophan radicals formed on horse metmyoglobin, also forms a Cys-110-centered thiyl radical that can also form a peroxyl radical. The tryptophanyl radicals react both with molecular oxygen and with the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS). The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) traps the Tyr-103 radicals and the Cys-110 thiyl radical of human myoglobin, and 2-methyl-2-nitrosopropane (MNP) traps all of the tyrosyl radicals. When excess H2O2 is used, DBNBS traps only a tyrosyl radical on horse myoglobin, but the detection of peroxyl radicals and the loss of tryptophan fluorescence support tryptophan oxidation under those conditions. Kinetic analysis of the formation of the various free radicals suggests that tryptophanyl radical and tyrosyl radical formation are independent events, and that formation of the Cys-110 thiyl radical on human myoglobin occurs via oxidation of the thiol group by the Tyr-103 phenoxyl radical. Peptide mapping studies of the radical adducts and direct EPR studies at low temperature and room temperature support the conclusions of the EPR spin trapping studies.     

Participation of tyrosine residues of myoglobin in the disproportionateness reaction of heme iron (II) and (IV)]

By means of the comparison of the constant oxidation reactions of both the myoglobin modified by N-acetylimidazole and the intact myoglobin in the presence of H2O2 or ferrylmyoglobin we characterized the role of the tyrosine residues (Tyr) of myoglobin in the synproportionation reaction between heme iron (II) of one molecule and heme iron (IV)--of another. It was demonstrated that Tyr derivatization resulted in the decrease of the velocity of redox interaction between deoxymyoglobin (II) and ferrylmyoglobin (IV) and led to the decrease of the efficiency of oxymyoglobin deoxygenation. The effects were shown to be independent on Tyr quantity in myoglobin molecule and to have the same character for both the sperm-whale myoglobin and the horse myoglobin.     

Effects of reactive species of oxygen and nitrogen on iron ion release from ferritin and synthesis of dinitrosyl iron complexes

Using EPR spectroscopy it was established that Fe ions released from ferritin under the action of glutathione and superoxide took part in the formation of dinitrosyl complexes of iron with glutathione (DNIC). The reaction between O2-. and NO resulted in the formation of peroxynitrite, which oxidized glutathione to the thiyl radical. In these conditions, DNIC did not inhibit the formation of thiyl radicals but effectively slowed down the oxidative destruction of beta-carotene by peroxynitrite and free radicals of lipids. In the presence of glutathione, the inversion of the antioxidant properties of DNIC into prooxidant ones took place. S-nitrosoglutathione prevented this inversion and suppressed the free-radical oxidation of beta-carotene induced by ferritin. It was proposed that the equilibrium between S-nitrosoglutathione, DNIC, "free Fe" ions and ferritin may determine the balance between prooxidant and antioxidant processes in living organisms.     

Determination of the chirality of the saturated pyrrole in sulfmyoglobin using the nuclear Overhauser effect

The interproton nuclear Overhauser effect (NOE) and paramagnetic dipolar relaxation rates for hyperfine-shifted resonances in the proton NMR spectra of sperm whale met-cyano sulfmyoglobin have led to the location and assignment of the proton signals of the heme pocket residue isoleucine 99 (FG5) in two sulfmyoglobin isomers. Dipolar relaxation rates of these protein signals indicate a highly conserved geometry of the heme pocket upon sulfmyoglobin formation, while the similar upfield direction of dipolar shifts for this residue to that observed in native sperm whale myoglobin reflects largely retained magnetic properties. Dipolar connectivity of this protein residue to the substituents of the reacted heme pyrrole ring B defines the stereochemistry of the puckered thiolene ring found in one isomer, with the 3-CH3 tilted out of the heme plane proximally. The chirality of the saturated carbons of pyrrole ring B in both the initial sulfmyoglobin product and the terminal alkaline product is consistent with a mechanism of formation in which an atom of sulfur is incorporated distally to form an episulfide across ring B, followed by reaction of the vinyl group to yield the thiolene ring that retains the C3 chirality.     

YajL, the prokaryotic homolog of the Parkinsonism-associated protein DJ-1, protects cells against protein sulfenylation

YajL is the closest Escherichia coli homolog of the Parkinsonism-associated protein DJ-1, a multifunctional oxidative stress response protein whose biochemical function remains unclear. We recently described the oxidative-stress-dependent aggregation of proteins in yajL mutants and the oxidative-stress-dependent formation of mixed disulfides between YajL and members of the thiol proteome. We report here that yajL mutants display increased protein sulfenic acids levels and that formation of mixed disulfides between YajL and its protein substrates in vivo is inhibited by the sulfenic acid reactant dimedone, suggesting that YajL preferentially forms disulfides with sulfenylated proteins. YajL (but not YajL(C106A)) also forms mixed disulfides in vitro with the sulfenylated form of bovine serum albumin. The YajL-serum albumin disulfides can be subsequently reduced by glutathione or dihydrolipoic acid. We also show that DJ-1 can form mixed disulfides with sulfenylated E. coli proteins and with sulfenylated serum albumin. These results suggest that YajL and possibly DJ-1 function as covalent chaperones involved in the detection of sulfenylated proteins by forming mixed disulfides with them and that these disulfides are subsequently reduced by low-molecular-weight thiols.     

Distribution of oxidized and reduced forms of glutathione and cysteine in rat plasma

The distribution of the glutathionyl moiety between reduced and oxidized forms in rat plasma was markedly different than that for the cysteinyl moiety. Most of the glutathionyl moiety was present as mixed disulfides with cysteine and protein whereas most of the cysteinyl moiety was present as cystine. Seventy percent of total glutathione equivalents was bound to proteins in disulfide linkage. The distribution of glutathione equivalents in the acid-soluble fraction was 28.0% as glutathione, 9.5% as glutathione disulfide, and 62.6% as the mixed disulfide with the cysteinyl moiety. In contrast, 23% of total cysteine equivalents was protein-bound. The distribution of cysteine equivalents in the acid-soluble fraction was 5.9% as cysteine, 83.1% as cystine, and 10.8% as the mixed disulfide with the glutathionyl moiety. A first-order decline in glutathione occurred upon in vitro incubation of plasma and was due to increased formation of mixed disulfides of glutathione with cysteine and protein. This indicates that plasma thiols and disulfides are not at equilibrium, but are in a steady-state maintained in part by transport of these compounds between tissues during the inter-organ phase of their metabolism. The large amounts of protein-bound glutathione and cysteine provide substantial buffering which must be considered in analysis of transient changes in glutathione and cysteine. In addition, this buffering may protect against transient thiol-disulfide redox changes which could affect the structure and activity of plasma and plasma membrane proteins.     

Inactivation of Ascorbate Peroxidase by Thiols Requires Hydrogen Peroxide

The hydrogen peroxide-dependent oxidation of ascorbate by ascorbateperoxidase from tea leaves was inhibited by thiols, such asdithiothreitol, glutathione, mercaptoethanol and cysteine. Thesethiols themselves did not inactivate the enzyme. However, theyinactivated the enzyme when hydrogen peroxide was produced bythe metal-catalyzed oxidation of thiols or when exogenous hydrogenperoxide was added. Thiols were oxidized by ascorbate peroxidaseand hydrogen peroxide to thiyl radicals, as detected by theESR spectra of the thiyl radical-5,5'-dimethyll- pyrroline-N-oxidieadducts. Inactivation of ascorbate peroxidase by thiols andhydrogen peroxide is caused by the interaction of the enzymewith the thiyl radicals produced at its reaction center. (Received September 10, 1991; Accepted December 9, 1991)     

A thin-gel isoelectric focusing method for quantitation of protein S-thiolation

A thin-gel isoelectric focusing method has been developed for analysis of protein S-thiolation (formation of mixed disulfides with low molecular weight thiols). The method is rapid and it can be used with 3 to 5 micrograms of a pure protein, or 15 to 20 micrograms of tissue extract protein. It is possible to detect a modification of the protein sulfhydryl by either charged or uncharged thiols, and to determine the quantity of different S-thiolated protein species in a modified sample. The method was used to quantitate the amount of S-thiolation of phosphorylase b in a reaction with oxidized glutathione that produced four S-thiolated forms of the enzyme. The method was also used to detect S-thiolation of two proteins in a cardiac tissue extract treated with diamide. One of the protein bands was shown to be S-thiolated with both cysteine and glutathione, while the other band was S-thiolated only with glutathione.     

Superoxide induces endothelial nitric-oxide synthase protein thiyl radical formation, a novel mechanism regulating eNOS function and coupling

An increase in production of reactive oxygen species resulting in a decrease in nitric oxide bioavailability in the endothelium contributes to many cardiovascular diseases, and these reactive oxygen species can oxidize cellular macromolecules. Protein thiols are critical reducing equivalents that maintain cellular redox state and are primary targets for oxidative modification. We demonstrate endothelial NOS (eNOS) oxidant-induced protein thiyl radical formation from tetrahydrobiopterin-free enzyme or following exposure to exogenous superoxide using immunoblotting, immunostaining, and mass spectrometry. Spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) followed by immunoblotting using an anti-DMPO antibody demonstrated the formation of eNOS protein radicals, which were abolished by superoxide dismutase and L-NAME, indicating that protein radical formation was due to superoxide generation from the eNOS heme. With tetrahydrobiopterin-reconstituted eNOS, eNOS protein radical formation was completely inhibited. Using mass spectrometric and mutagenesis analysis, we identified Cys-908 as the residue involved in protein radical formation. Mutagenesis of this key cysteine to alanine abolished eNOS thiyl radical formation and uncoupled eNOS, leading to increased superoxide generation. Protein thiyl radical formation leads to oxidation or modification of cysteine with either disulfide bond formation or S-glutathionylation, which induces eNOS uncoupling. Furthermore, in endothelial cells treated with menadione to trigger cellular superoxide generation, eNOS protein radical formation, as visualized with confocal microscopy, was increased, and these results were confirmed by immunoprecipitation with anti-eNOS antibody, followed by immunoblotting with an anti-DMPO antibody. Thus, eNOS protein radical formation provides the basis for a mechanism of superoxide-directed regulation of eNOS, involving thiol oxidation, defining a unique pathway for the redox regulation of cardiovascular function.     

Hypochlorite-induced oxidation of thiols: Formation of thiyl radicals and the role of sulfenyl chlorides as intermediates

Activated phagocytic cells generate hypochlorite (HOCl) via release of hydrogen peroxide and the enzyme myeloperoxidase. HOCl plays an important role in bacterial cell killing, but excessive or misplaced production of HOCl is also known to cause tissue damage. Studies have shown that low-molecular-weight thiols such as reduced glutathione (GSH), and sulfur-containing amino acids in proteins, are major targets for HOCl. Radicals have not generally been implicated as intermediates in thiol oxidation by HOCl, though there is considerable literature evidence for the involvement of radicals in the metal ion-, thermal- or UV light-catalysed decomposition of sulfenyl or sulfonyl chlorides which are postulated intermediates in thiol oxidation. In this study we show that thiyl radicals are generated on reaction of a number of low-molecular-weight thiols with HOCl. With sub-stoichiometric amounts of HOCl, relative to the thiol, thiyl radicals are the major species detected by EPR spin trapping. When the HOCl is present in excess over the thiol, additional radicals are detected with compounds which contain amine functions; these additional radicals are assigned to nitrogen-centered species. Evidence is presented for the involvement of sulfenyl chlorides (RSCl) in the formation of these radicals, and studies with an authentic sulfenyl chloride have demonstrated that this compound readily decomposes in thermal-, metal-ion- or light-catalysed reactions to give thiyl radicals. The formation of thiyl radicals on oxidation of thiols with HOCl appears to compete with non-radical reactions. The circumstances under which radical formation may be important are discussed.     

Cigarette smoke induced oxidation of human plasma proteins,lipids, and antioxidants; selective protection by the biothiols dihydrolipoic acid and glutathione

Summary

Exposure of human plasma to gas-phase cigarette smoke (CS) causes loss of human plasma antioxidants, protein modification (Frei et al, Biochem J, 1991 277:133–138; Reznick et al, Biochem J, 1992 286: 607–611) and a minimal amount of lipid oxidation. Ascorbic acid was found to prevent CS-induced lipid peroxidation and glutathione (GSH) partially protected against protein modification, as determined by loss of protein -SH groups and by increases in carbonyl content as a measure of protein oxidation. In the present study we demonstrate that dihydrolipoic acid (0.25–1.0 mM) decreases CS-induced protein carbonyls, α-tocopherol loss, and lipid hydroperoxide formation in plasma. In contrast GSH (1 mM) failed to influence CS-induced loss of α-tocopherol, and was 50% as effective as dihydrolipoate in protecting against CS-induced protein carbonyl formation. On the other hand, lipoic acid (oxidized form of dihydrolipoic acid) and oxidized glutathione (GSSG) had minimal effect in protecting against the CS-induced protein modifications. These findings demonstrate that low molecular weight thiols are capable of modifying the effect of gas-phase CS on biological fluids. Dihydrolipoate appears to be particularly useful in that it was shown to conserve ascorbic acid and α-tocopherol, i.e. supporting the antioxidant network concept in protection against protein and lipid oxidation.     

Interactions of the antitumor drug, etoposide, with reduced thiols in vitro and in vivo

The interaction of activated etoposide, 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyra noside) (VP-16), with thiols has been studied both in vitro and in vivo in mice. We have found that both glutathione (GSH) and cysteine rapidly reduce the VP-16 free radical, which results in the regeneration of the parent drug and the oxidation of the thiol. Using spin-trapping and electron spin resonance (ESR) techniques, we have shown that this one-electron/hydrogen donation by thiols forms thiyl radicals (RS.) which are intermediates for the formation of the oxidized thiols. The administration of VP-16 in vivo to mice decreased the total thiol levels in liver and concomitantly increased the formation of oxidized thiols. Furthermore, VP-16 stimulated glutathione reductase in liver. While administration of VP-16 also increased the total thiol pools in kidney, in contrast, no significant effects were observed on lung and heart thiol pools.     

Electron paramagnetic resonance spectroscopy as a probe of coordination structure in green heme systems: iron chlorins and iron formylporphyrins reconstituted into myoglobin

A series of ferric low-spin derivatives of myoglobin containing its natural prosthetic group, iron protoporphyrin IX, and reconstituted with iron heme s (a formyl-substituted porphyrin) and iron methylchlorin have been examined using low-temperature electron paramagnetic resonance (EPR) spectroscopy. Good agreement is observed between the EPR properties of parallel derivatives of natural myoglobin and heme s-myoglobin. Likewise, the EPR properties of parallel adducts of three types of iron chlorins, methylchlorin-myoglobin, sulfyomyoglobin (a myoglobin derivative known to contain a chlorin macrocycle) and synthetic chlorin models are similar to each other. The ferric chlorin systems are shown to exhibit increased tetragonality and decreased rhombicity values relative to protoporphyrin/formylporphyrin systems. Thus, EPR spectroscopy is a very useful technique with which to probe the coordination structure of naturally occurring iron chlorin proteins and the method can be used to distinguish between proteins containing iron formylporphyrins and iron chlorin prosthetic groups.