Identification of a copper-sensitive ascorbate peroxidase in the unicellular green alga Selenastrum capricornutum

Extracts from the unicellular green alga Selenastrum capricornutum exhibit high superoxide dismutase activity, but only traces of catalase activity. The excess hydrogen peroxide (HO) generated by the superoxide dismutase in S. capricornutum may be degraded by a unique peroxidase. This peroxidase has a high specificity for ascorbate as its electron donor. The enzyme has an optimum pH at 8, is insensitive to cyanide and is inhibited by oxine. Addition of low concentrations of copper to algal cultures stimulates the peroxidase activity threefold. This enzymatic system could be used as a sensitive bioindicator for copper in fresh water.     

Characterization of an iron- and manganese-containing superoxide dismutase from Methylobacillus sp. strain SK1 DSM 8269

A superoxide dismutase was purified 62-fold in seven steps to homogeneity from Methylobacillus sp. strain SK1, an obligate methanol-oxidizing bacterium, with a yield of 9.6%. The final specific activity was 4,831 units per milligram protein as determined by an assay based on a 50% decrease in the rate of cytochrome c reduction. The molecular weight of the native enzyme was estimated to be 44,000. Sodium dodecyl sulfate gel electrophoresis revealed two identical subunits of molecular weight 23,100. The isoelectric point of the purified enzyme was found to be 4.4. Maximum activity of the enzyme was measured at pH 8. The enzyme was stable at pH range from 6 to 8 and at high temperature. The enzyme showed an absorption peak at 280 nm with a shoulder at 292 nm. Hydrogen peroxide and sodium azide, but not sodium cyanide, was found to inhibit the purified enzyme. The enzyme activity in cell-free extracts prepared from cells grown in manganese-rich medium, however, was not inhibited by hydrogen peroxide but inhibited by sodium azide. The activity in cell extracts from cells grown in iron-rich medium was found to be highly sensitive to hydrogen peroxide and sodium azide. One mol of native enzyme was found to contain 1.1 g-atom of iron and 0.7 g-atom of manganese. The N-terminal amino acid sequence of the purified enzyme was Ala-Tyr-Thr-Leu-Pro-Pro-Leu-Asn-Tyr-Ala-Tyr. The superoxide dismutase of Methylobacillus sp. strain SK1 was found to have antigenic sites identical to those of Methylobacillus glycogenes enzyme. The enzyme, however, shared no antigenic sites with Mycobacterium sp. strain JC1, Methylovorus sp. strain SS1, Methylobacterium sp. strain SY1, and Methylosinus trichosproium enzymes.     

Cobalt bovine superoxide dismutase. Reactivity of the cobalt chromophore in the copper-containing and in the copper-free enzyme.

1. The reactivity of the zinc site of bovine superoxide dismutase has been probed by observing optical and electron paramagnetic resonance changes, under several conditions, of the Co(II)-substituted protein. 2. Only in the absence of copper are the optical and electron paramagnetic resonance spectra of the cobalt chromophore appreciably affected by alkaline pH or by cyanide. With both reagents the reaction with the copper-containing protein appears to involve the water molecule bound to the copper and does not affect the magnetic coupling between copper and cobalt. 3. The reaction of cyanide with the copper-free Co(II) protein leads to a slow detachment of cobalt from the protein as pentacyanocobalt. An oxygen adduct forms in air, analogous to that described in Co(II) carbonic anhydrase (Haffner, P. H. and Coleman, J. E. (1975) J. Biol. Chem. 250, 996--1005.) 4. Acid titration modifies the Co(II) spectra in the same way in the Cu-containing and in the Cu-free protein and brings about uncoupling of the Co(II)--Cu(II) system. Protonation of histidine-61 on the zinc facing nitrogen is suggested. 5. H2O2 modifies the cobalt chromophore only in the presence of copper. Magnetic coupling between Cu(II) and Co(II) seems to be still present after H2O2 inactivation of the enzyme.     

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides

Aspects of the utilization of copper by the fungus, Dactylium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, and extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (holoenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (less than 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 micrometer, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 micrometer medium copper, holoenzyme secretion is maintained throughout cell growth. The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN(-)-insensitive, manganese form of this enzyme. Cells grown at 10 micrometer copper show 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.     

Microbial oxidation of amines. Spectral and kinetic properties of the primary amine dehydrogenase of Pseudomonas AM 1

1. An improved procedure is reported for purification of the amine dehydrogenase from methylamine-grown Pseudomonas AM1 which yielded a product homogeneous by sedimentation and disc-electrophoretic analysis, with molecular weight of 133000. 2. The purified enzyme had absorption maxima at 280 and 430nm. On aging, a third peak appeared at 325nm, and the 430nm peak decreased in intensity. This spectrum was independent of pH. 3. Addition of 2.5mm-semicarbazide, phenylhydrazine, hydrazine or hydroxylamine produced modified spectra with maxima respectively at 400, 440, 395 and 425nm. 4. Aerobic addition of methylamine resulted in a bleaching of the 430nm peak and the appearance of a new one at 325nm. This spectral change was retained after removal of the methylamine by dialysis. The original spectrum could be restored on addition of phenazine methosulphate. 5. Addition of borohydride partially inactivated the enzyme and produced spectral changes similar to those observed with methylamine. Pre-treatment with methylamine prevented the inactivation by borohydride. The degree of inactivation could be increased by alternate phenazine methosulphate and borohydride treatments. 6. The addition of methylamine or borohydride each caused shifts in the fluorescence emission maximum from 348 to 380nm. 7. Lineweaver-Burk plots of reciprocal activity against reciprocal concentration of either of the substrates n-butylamine or phenazine methosulphate were consistent with a mechanism that involves interconversion of two free forms of the enzyme by the two substrates. 8. The enzyme, although spectrally modified, was not inactivated by dialysis against diethyldithiocarbamate, and contained about 0.27 g-atom of copper/mol, with small traces of cobalt, iron and zinc. 9. Conventional methods of resolution did not release the prosthetic group. Heat denaturation after treatment of the enzyme with methylamine liberated a yellow chromophore which did not reactivate resolved aspartate aminotransferase, and whose spectral, electrophoretic and fluorescence properties did not agree with any recognizable pyridoxal derivatives. 10. Despite the inconclusive results with the isolated chromophore, the observations on the enzyme suggest that it may contain a pyridoxal derivative bound as a Schiff's base which is converted into the pyridoxamine form on aerobic treatment with methylamine and reconverted into the pyridoxal form with phenazine methosulphate. 11. The copper detected is probably not involved in the enzyme mechanism, since most copper-chelating agents are not inhibitory, and since the enzyme does not react with oxygen.     

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides

Aspects of the utilization of copper by the fungus, Dactytium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, an extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (haloenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (< 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 μM, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 μM medium copper, holoenzyme secretion is maintained throughout cell growth.The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN^?-insensitive, manganese form of this enzyme. Cells grown at 10 μM copper shown 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.     

Functional expression of a recombinant copper/zinc superoxide dismutase of filarial nematode, Brugia malayi

A gene encoding a copper/zinc superoxide dismutase (Cu/ Zn-SOD) of a filarial nematode, Brugia malayi, has been isolated and the biochemical properties of a functionally expressed recombinant enzyme were investigated. The cloned complementary DNA contained a single open reading frame of 477 bp encoding 158 amino acids (aa), which conserved metal-binding residues as well as residues specific for Cu/Zn-SODs. Comparison of the deduced aa sequence of the enzyme with that of other helminthes species, including filarial worms, exhibited high degree of similarities (49-98%). Recombinant enzyme of 32 kDa had an isoelectric point of 6.6 and was shown to consist of 2 subunits linked by interchain disulfide bonds. Enzyme activity of the recombinant protein was inhibited by potassium cyanide and hydrogen peroxide but not by sodium azide. It showed a wide range of pH optima, i.e., 7.0-11.0 and was highly resistant to heat inactivation.     

Characterization of superoxide dismutase from south Indian scorpion venom.

A manganese containing superoxide dismutase was purified to homogeneity from the venom of scorpion Heterometrus fulvipes by ammonium sulfate fractionation followed by gel filtration on Sephadex G-100 and ion exchange chromatography on DEAE-cellulose. The enzyme has a molecular weight of 100,000. Optimum pH for enzyme activity was 8.5 and optimum temperature was 45 degrees C. The enzyme was not sensitive to either cyanide or hydrogen peroxide but was inhibited by chloroform-ethanol mixture and p-hydroxymercuribenzoate. Metal chelators, EDTA, o-phenanthroline and diethyldithiocarbamate inhibited the enzyme activity in decreasing order. The effect of 6 M urea, sodium dodecylsulfate, guanidinium chloride and nitroprusside on enzyme activity has been studied. An antiserum raised against H. fulvipes venom inhibited the superoxide dismutase activity.     

The role of copper atoms in the cytochrome oxidase reaction]

It was found that cytochrome oxidase from bovine cardiac muscle possesses marked superoxide dismutase activity. Superoxide dismutase activity is inhibited by cyanide and azide or by alkaline or thermal treatments. This activity is also suppressed by chelating agents, e.g. bathocuproin. The data obtained indicate that superoxide dismutase activity of cytochrome oxidase is due to the copper atoms of the enzyme. The experiments on the copper-containing subunit support this conclusion. Possible physiological significance of superoxide dismutase activity of cytochrome oxidase is discussed.     

用固定化方法研究超氧化物歧化酶的活性与结构的关系

 采用吸附、包埋、共价交联等方法固定化超氧化物歧化酶(SOD)所得固定化酶活力回收都不高,表明酶的催化反应速率受超氧阴离子自由基(O_2~-)扩散速率的控制。用海藻酸钠包埋SOD,固定化酶不表现活力,破固定化后所得的糊状物却有很高的活力。用戊二醛交联所得的固定化酶活力回收率也很低,表明ε-NH_3~+的正电荷是酶活力所必需。     

Isolation of Assimilatory- and Dissimilatory-Type Sulfite Reductases from Desulfovibrio vulgaris

Bisulfite reductase (desulfoviridin) and an assimilatory sulfite reductase have been purified from extracts of Desulfovibrio vulgaris. The bisulfite reductase has absorption maxima at 628, 580, 408, 390, and 279 nm, and a molecular weight of 226,000 by sedimentation equilibrium, and was judged to be free of other proteins by disk electrophoresis and ultracentrifugation. On gels, purified bisulfite reductase exhibited two green bands which coincided with activity and protein. The enzyme appears to be a tetramer but was shown to have two different types of subunits having molecular weights of 42,000 and 50,000. The chromophore did not form an alkaline ferrohemochromogen, was not reduced with dithionite or borohydride, and did not form a spectrally visible complex with CO. The assimilatory sulfite reductase has absorption maxima at 590, 545, 405 and 275 nm and a molecular weight of 26,800, and appears to consist of a single polypeptide chain as it is not dissociated into subunits by sodium dodecyl sulfate. By disk electrophoresis, purified sulfite reductase exhibited a single greenish-brown band which coincided with activity and protein. The sole product of the reduction was sulfide, and the chromophore was reduced by borohydride in the presence of sulfite. Carbon monoxide reacted with the reduced chromophore but it did not form a typical pyridine ferrohemochromogen. Thiosulfate, trithionate, and tetrathionate were not reduced by either enzyme preparation. In the presence of 8 M urea, the spectrum of bisulfite reductase resembles that of the sulfite reductase, thus suggesting a chemical relationship between the two chromophores.     

Superoxide dismutase isoenzymes in bovine and human milk

The presence of superoxide dismutase in bovine and human milk was investigated by ultrafiltration, gel filtration, and isoelectric focusing. Conclusive evidence for the presence of this enzyme in both milks is presented. The molecular weight of the enzyme was estimated by gel filtration on Sephadex G-100 to be 30,000, which is consistent with reported values for the copper, zinc form of superoxide dismutase. In addition, enzyme activity was inhibited by cyanide, thus eliminating the possibility that the enzyme was present in the manganese form. Several isoenzymes were detected by isoelectric focusing in polyacrylamide gel, and the isoenzyme pattern in bovine milk was the same as that found for bovine plasma, suggesting that milk superoxide dismutase originates from plasma. It may be that the presence of copper, zinc superoxide dismutase in milk is important for the maintenance of its oxidative stability.     

Metal sites of copper-zinc superoxide dismutase.

Silver-copper and silver-cobalt proteins have been prepared in which Ag+ resides in the native copper site of superoxide dismutase and either Cu2+ of Co2+ reside in the zinc site. The electron paramagnetic resonance (EPR) spectrum of the copper and the visible absorption spectrum of the cobalt greatly resemble those of either Cu4 of Cu2,Cu2,Co2 proteins, respectively, in which the copper of the native copper sites has been reduced. It was found that, unlike cyanide, azide anion would not perturb the EPR spectrum of Ag2,Cu2 protein. Since azide produces the same perturbation upon the EPR spectrum of native and Cu2 proteins, it must bind to the copper and not the zinc of superoxide dismutase. A model of the metal sites of the enzyme has been fitted to a 3-A electron-density map using an interactive molecular graphics display. The model shows that histidine-61, which appears to bind both copper and zinc, does not lie in the plane of the copper and its three other histidine ligands, but occupies a position intermediate between planar and axial. This feature probably accounts for the rhombicity of the EPR spectrum and the activity of the enzyme.     

Photochemical and chemical studies on the chromophore of bacteriorhodopsin.

The chromophore (purple complex) of bacteriorhodopsin is reduced by sodium borohydride upon illumination to RPhv with a three-peaked absorption band at 360 nm. Treatment of this reduction product with ultraviolet light or acid yields a modified product from which retro-retinyllysine can be obtained by alkaline hydrolysis. No reduction of the 412 nm complex was found. Under specific conditions the purple complex equilibrates with a photochemically active 460 nm form that can be reduced by borohydride in the dark. This reduction product RP460 behaves idential to RPHV. Reconstitution of the purple complex from chromophore-free membrane (apomembrane) and retinal occurs via intermediates. The first (lambdamax 400nm) shows a three-peaked absorption band and is reduced to RP400 without a change of the three-peaked absorption (lambdamax 360 nm). The same product is obtained from apomembrane and retinol. Detergents shift the absorption band to 330 nm in all cases. From the experiments described no participation of retro-retinal structures during the photochemical cycle can be concluded but stereospecific interaction of the retinal moiety with the protein resulting in a specific retinal conformation os omdocated by the spectral changes observed.     

The ionization states of the 5'-phosphate group in the various coenzyme forms bound to mitochondrial aspartate aminotransferase

We have carried out a Fourier transform infrared spectroscopic study of mitochondrial aspartate aminotransferase in the spectral region where phosphate monoesters give rise to absorption. Infrared spectra in the above-mentioned region are dominated by protein absorption. Yet, below 1020 cm-1 protein interferences are minor, permitting the detection of the band arising from the symmetric stretching of dianionic phosphate monoesters [T. Shimanouchi, M. Tsuboi, and Y. Kyogoku (1964) Adv. Chem. Phys. 8, 435-498]. The integrated intensity of this band in several enzyme forms (pyridoxal phosphate, pyridoxamine phosphate, and sodium borohydride-reduced, pyridoxyl phosphate form) does not change with pH in the range 5-9. This behavior contrasts that of free pyridoxal phosphate (PLP) and pyridoxamine phosphate (PMP) in solution, where the dependence of the same infrared band intensity with pH can be correlated to the known pK values for the 5'-phosphate ester in solution. The integrated intensity value of this infrared band for the PLP enzyme form before and after reduction with sodium borohydride is close to that given by free PLP at pH 8-9. These results are taken as evidence that in the active site of mitochondrial aspartate aminotransferase the 5'-phosphate group of PLP remains mostly dianionic even at a pH near 5. Thus, it is suggested that the chemical shift changes associated with pH titrations of various PLP forms reported in a previous 31P NMR study of this enzyme [M. E. Mattingly, J. R. Mattingly, and M. Martinez-Carrion (1982) J. Biol. Chem. 257, 8872] are due to the fact that the phosphorus chemical shift senses the O-P-O bond distortions induced by the ionization of a nearby residue. Since no chemical shift changes were observed in pH titrations of the PMP forms (lacking an ionizable internal aldimine) of this isozyme, the Schiff base between PLP and Lys-258 at the active site is the most likely candidate for the ionizing group influencing the phosphorus chemical shift in this enzyme.     

人胎肝超氧化物歧化酶的研究

采用离子交换和凝胶过滤层析法,从正常人胎肝提取、纯化铜锌超氧化物歧化酶(Cu.Zn-SOD),并对其性质作了研究,结果测得人胎肝组织Cu·Zn-SOD平均含量为6.44unit/g湿重,并发现随着胎龄的增加人胎肝Cu.Zn-SOD含量上升;纯化后的Cu·Zn-SOD在聚丙烯酰胺凝胶电泳图谱上呈现单一区带;其活性受氰化钾抑制;以原子吸收光谱测得其铜、锌含量分别为0.39％和0.1％,采用SDS-聚丙烯酰胺凝胶电泳测得其亚基分子量为16kD;氨基酸自动分析仪测得其亚基的氨基酸残基数为151.5;在紫外吸收光谱上于265nm和258nm处分别出现两个吸收高峰。本研究结果表明,人胎肝Cu.Zn-SOD与成人肝及其他组织的Cu.Zn-SOD理化性质上基本一致。     

Purification, N-Terminal Amino Acid Sequence and Partial Characterization of A Cu,Zn Superoxide Dismutase From the Pathogenic Fungus Aspergillusfumiga Tus

A superoxide dismutase (SOD) has been purified to homogeneity from the fungal pathogen Aspergillus fumigatus using a combination of cell homogenization, isoelectric focusing and gel filtration FPLC. The N-terminal amino acid sequence of the purified enzyme demonstrated substantial homology to known Cu, Zn superoxide dismutases for a range of organisms, including Neurospora crassa and Saccharomyces cerevisiae. The enzyme subunit has a pl of 5.9, a relative molecular mass of 19 kDa and a spectral absorbance maximum of 550nm. The non reduced enzyme has a relative molecular mass of 95 kDa. The enzyme remained active after prolonged incubation at 70°C and was pH insensitive in the range 7-11. Potassium cyanide and diethyldithiocarbamate, known Cu, Zn SOD inhibitors, caused inhibition of the purified enzyme at working concentrations of 0.25 mM, whilst sodium azide and o-phenanthroline demonstrated inhibition at higher concentrations (10-30 mM). SOD activity was also detectable in culture filtrate of A. fumigatus. This enzyme may have a potential role as a virulence factor in the avoidance of neutrophil and phagocyte oxidative burst killing mechanisms.     

Purification and characterization of glutamate decarboxylase from Solanum tuberosum

Glutamate decarboxylase has been purified from potato tubers. The final preparation was homogeneous as judged from native and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Gel filtration on Sephadex G-200 gave a relative molecular mass Mr, of 91 000 for the native enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a subunit Mr of 43 000. Thus the enzyme appears to be a dimer of identical subunits. It has 2 mol pyridoxal 5'-phosphate/mol protein, which could not be removed by exhaustive dialysis or gel filtration on Sephadex G-25. The enzyme has an absorption maximum at 370 nm in sodium phosphate buffer, pH 5.8. Reduction of the enzyme with sodium borohydride abolished the absorption maximum at 370 nm with attendant loss of catalytic activity. The enzyme exhibited pH-dependent spectral changes. The enzyme was specific for L-glutamate and could not decarboxylate other amino acids tested. The enzyme was maximally active at pH 5.8 and a temperature of 37 degrees C. Isoelectric focussing gave a pI of 4.7 Km values for L-glutamate and pyridoxal 5'-phosphate were 5.6 mM and 2 microM respectively. Thiol-directed reagents and heavy metal ions inhibited the enzyme, indicating that an -SH group is required for activity. The nature of the functional groups at the active site of the enzyme was inferred from competitive inhibition studies. L-Glutamate promoted inactivation of the enzyme caused by decarboxylation-dependent transamination was demonstrated. The characteristics of potato enzyme were compared with enzyme from other sources.     

Photochemically modified myeloperoxidase, with optical spectral properties analogous to those of lactoperoxidase, retains its original catalytic activity

During the course of a reducing reaction using ketyl radicals generated from ketone photoreduction with ultraviolet light, a photoinduced chemical modification of the chromophore group in myeloperoxidase has been found. Light absorption and resonance Raman spectra for this modified enzyme indicated an iron porphyrin chromophore group. The alkaline pyridine hemochrome of the modified enzyme exhibited an optical spectrum closely related to that of iron protoporphyrin IX. The chromophore group of the modified myeloperoxidase was cleaved from the protein by methoxide. Proton magnetic resonance of the diamagnetic bis(cyanide) compound of the extracted heme group showed the presence of two vinyl and three methyl side chains associated with a porphyrin macrocycle. These data provide further insight into the structure of the active site in myeloperoxidase. The EPR spectral properties and enzymatic activities of the native myeloperoxidase are essentially conserved in the modified enzyme. Our present results indicate that the heme peripheral substituent is modified while the stereochemical structure surrounding the chromophore group is not altered by the photochemical modification.