Novel oligosaccharide has suppressive activity against human leukemia cell proliferation.

Various oligosaccharides containing galactose(s) and one glucosamine (or N-acetylglucosamine) residues with beta1-4, alpha1-6 and beta1-6 glycosidic bond were synthesized; Galbeta1-4GlcNH(2), Galalpha1-6GlcNH(2), Galalpha1-6GlcNAc, Galbeta1-6GlcNH(2), Galbeta1-4Galbeta1-4GlcNH(2) and Galbeta1-4Galbeta1-4GlcNAc. Galalpha1-6GlcNH(2) (MelNH(2)) and glucosamine (GlcNH(2)) had a suppressive effect on the proliferation of K562 cells, but none of the other saccharides tested containing GlcNAc showed this effect. On the other hand, the proliferation of the human normal umbilical cord fibroblast was suppressed by none of the saccharides other than GlcNH(2). Adding Galalpha1-6GlcNH(2) or glucosamine to the culture of K562 cell, the cell number decreased strikingly after 72 h. Staining the remaining cells with Cellstain Hoechst 33258, chromatin aggregation was found in many cells, indicating the occurrence of cell death. Furthermore, all of the cells were stained with Galalpha1-6GlcNH-FITC (MelNH-FITC). Neither the control cells nor the cells incubated with glucosamine were stained. On the other hand, when GlcNH-FITC was also added to cell cultures, some of them incubated with Galalpha1-6GlcNH(2) were stained. The difference in the stainability of the K562 cells by Galalpha1-6GlcNH-FITC and GlcNH-FITC suggests that the intake of Galalpha1-6GlcNH(2) and the cell death induced by this saccharide is not same as those of glucosamine. The isolation of the Galalpha1-6GlcNH(2) binding protein was performed by affinity chromatography (melibiose-agarose) and LC-MS/MS, and we identified the human heterogeneous ribonucleoprotein (hnRNP) A1 (34.3 kDa) isoform protein (30.8 kDa). The hnRNP A1 protein was also detected from the eluate(s) of the MelNH-agarose column by the immunological method (anti-hnRNP-A1 and HRP-labeled anti-mouse IgG (gamma) antibodies).

Characterization and cloning of an extremely thermostable, Pyrococcus furiosus-type 4Fe ferredoxin from Thermococcus profundus.

An extremely thermostable [4Fe-4S] ferredoxin was isolated under anaerobic conditions from a hyperthermophilic archaeon Thermococcus profundus, and the ferredoxin gene was cloned and sequenced. The nucleotide sequence of the ferredoxin gene shows the ferredoxin to comprise 62 amino acid residues with a sequence similar to those of many bacterial and archaeal 4Fe (3Fe) ferredoxins. The unusual Fe-S cluster type, which was identified in the resonance Raman and EPR spectra, has three cysteines and one aspartate as the cluster ligands, as in the Pyrococcus furiosus 4Fe ferredoxin. Under aerobic conditions, a ferredoxin was purified that contains a [3Fe-4S] cluster as the major Fe-S cluster and a small amount of the [4Fe-4S] cluster. Its N-terminal amino acid sequence is the same as that of the anaerobically-purified ferredoxin up to the 26th residue. These results indicate that the 4Fe ferredoxin was degraded to 3Fe ferredoxin during aerobic purification. The aerobically-purified ferredoxin was reversibly converted back to the [4Fe-4S] ferredoxin by the addition of ferrous ions under reducing conditions. The anaerobically-purified [4Fe-4S] ferredoxin is quite stable; little degradtion was observed over 20 h at 100 degrees C, while the half-life of the aerobically-purified ferredoxin is 10 h at 100 degrees C. Both the anaerobically- and aerobically-purified ferredoxins were found to function as electron acceptors for the pyruvate-ferredoxin oxidoreductase purified from the same archaeon.

Modification of a single tryptophan residue in human Cu,Zn-superoxide dismutase by peroxynitrite in the presence of bicarbonate.

Human recombinant Cu,Zn-SOD was reacted with peroxynitrite in a reaction mixture containing 150 mM potassium phosphate buffer (pH 7.4) 25 mM sodium bicarbonate, and 0.1 mM diethylenetriamine pentaacetic acid. Disappearance of fluorescence emission at 350 nm, which could be attributed to modification of a single tryptophan residue, was observed in the modified enzyme with a pH optimum of around 8.4. A fluorescence decrease with the same pH optimum was also observed without sodium bicarbonate, but with less efficiency. Amino acid contents of the modified enzyme showed no significant difference in all amino acids except the loss of a single tryptophan residue of the enzyme. The peroxynitrite-modified enzyme showed an increase in optical absorption around 350 nm and 30% reduced enzyme activity based on the copper contents. The modified enzyme showed the same electron paramagnetic resonance spectrum as that of the control enzyme. The modified Cu,Zn-SOD showed a single protein band in sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS--PAGE) and five protein bands in non-denaturing PAGE. From this evidence, we conclude that nitration and/or oxidation of the single tryptophan 32 and partial inactivation of the enzyme activity of Cu,Zn-SOD is caused by a peroxynitrite-carbon dioxide adduct without perturbation of the active site copper integrity.

Crystal structure of cambialistic superoxide dismutase from porphyromonas gingivalis.

The crystal structure of cambialistic superoxide dismutase (SOD) from Porphyromonas gingivalis, which exhibits full activity with either Fe or Mn at the active site, has been determined at 1.8-A resolution by molecular replacement and refined to a crystallographic R factor of 17.9% (Rfree 22.3%). The crystals belong to the space group P212121 (a = 75.5 A, b = 102.7 A, c = 99.6 A) with four identical subunits in the asymmetric unit. Each pair of subunits forms a compact dimer, but not a tetramer, with 222 point symmetry. Each subunit has 191 amino-acid residues most of which are visible in electron density maps, and consists of seven alpha helices and one three-stranded antiparallel beta sheet. The metal ion, a 3 : 1 mixture of Fe and Mn, is coordinated with five ligands (His27, His74, His161, Asp157, and water) arranged at the vertices of a trigonal bipyramid. Although the overall structural features, including the metal coordination geometry, are similar to those found in other single-metal containing SODs, P. gingivalis SOD more closely resembles the dimeric Fe-SODs from Escherichia coli rather than another cambialistic SOD from Propionibacterium shermanii, which itself is rather similar to other tetrameric SODs.

A change of the metal-specific activity of a cambialistic superoxide dismutase from Porphyromonas gingivalis by a double mutation of Gln-70 to Gly and Ala-142 to Gln.

Gln-70, which is located near the active-site metal, is conserved in aligned amino acid sequences of iron-containing superoxide dimutases (Fe-SODs) and cambialistic SOD from Porphyromonas gingivalis, but is complementarily substituted with Gln-142 in manganese-containing SODs (Mn-SODs). In order to clarify the contribution of this exchange of Gln to the metal-specific activity of P. gingivalis SOD, we have prepared a mutant of the enzyme with conversions of Gln-70 to Gly and Ala-142 to Gln. The ratio of the specific activities of Mn- to Fe-reconstituted P. gingivalis SOD increased from 1.4 in the wild-type to 3.5 in the mutant SODs. Furthermore, the visible absorption spectra of the Mn- and Fe-reconstituted mutant SODs more closely resembled that of Mn-specific SOD than that of the wild-type SOD. We conclude that a difference in configuration of the Gln residues of P. gingivalis SOD partially accounts for the metal-specific activity of the enzyme.

EPR and ligand field studies of iron superoxide dismutases and iron-substituted manganese superoxide dismutases: relationships between electronic structure of the active site and activity.

The problem of metal selectivity of iron/manganese superoxide dismutases (SODs) is addressed through the electronic structures of active sites using electron paramagnetic resonance and ligand field calculations. Studies of wild-type iron(III) SOD (FeSOD) from Escherichia coli and from Methanobacterium thermoautotrophicum and iron-substituted manganese(III) SOD (Fe(sub)MnSOD) from E. coli and from Serratia marcescens are reported. EPR spectroscopy of wild-type enzymes shows transitions within all three Kramers doublets identified by their g values. From the temperature dependence of the observed transitions, the zero-field splitting is found to be negative, D = -2 +/- 0.2 cm-1. The electronic structure is typical of a distorted trigonal bipyramid, all the EPR features being reproduced by ligand field analysis. This unique and necessary electronic structure characterizes wild-type enzymes whatever their classification from the amino acid sequence into iron or manganese types, as E. coli FeSOD or M. thermoautotrophicum FeSOD. In iron-substituted manganese SODs, reduced catalytic activity is found. We describe how inhomogeneity of all reported substituted MnSODs might explain the activity decrease. EPR spectra of substituted enzymes show several overlapping components. From simulation of these spectra, one component is identified which shares the same electronic structure of the wild-type FeSODs, with the proportion depending on pH. Ligand field calculations were performed to investigate distortions of the active site geometry which induce variation of the excitation energy of the lowest quartet state. The corresponding coupling between the ground state and the excited state is found to be maximum in the geometry of the native SODs. We conjecture that such coupling should be considered in the electron-transfer process and in the contribution of the typical electronic structure of FeSOD to the activity.

Inactivation of human manganese-superoxide dismutase by peroxynitrite is caused by exclusive nitration of tyrosine 34 to 3-nitrotyrosine.

Peroxynitrite has recently been implicated in the inactivation of many enzymes. However, little has been reported on the structural basis of the inactivation reaction. This study proposes that nitration of a specific tyrosine residue is responsible for inactivation of recombinant human mitochondrial manganese-superoxide dismutase (Mn-SOD) by peroxynitrite. Mass spectroscopic analysis of the peroxynitrite-inactivated Mn-SOD showed an increased molecular mass because of a single nitro group substituted onto a tyrosine residue. Single peptides that had different elution positions between samples from the native and peroxynitrite-inactivated Mn-SOD on reverse-phase high performance liquid chromatography were isolated after successive digestion of the samples by staphylococcal serine protease and lysylendopeptidase and subjected to amino acid sequence and molecular mass analyses. We found that tyrosine 34 of the enzyme was exclusively nitrated to 3-nitrotyrosine by peroxynitrite. This residue is located near manganese and in a substrate O-2 gateway in Mn-SOD.

Inactivation and destruction of conserved Trp159 of Fe-superoxide dismutase from Porphyromonas gingivalis by hydrogen peroxide.

The superoxide dismutase (SOD) of Porphyromonas gingivalis, an obligate anaerobe, was purified from Escherichia coli (sodA sodB mutant) harboring the P. gingivalis SOD-encoding gene. The purified protein contained both iron and a small amount of manganese. Iron- and manganese-reconstituted SOD, which contained one of these metals exclusively, showed specific activities of 1000 and 1200 U/mg/mol of metals/subunit, respectively. These values were similar to the specific activity of the native enzyme purified from the recombinant E. coli strain. The Fe-reconstituted enzyme was inactivated by 10 mM hydrogen peroxide to about 5% of its original activity after a 15 min incubation at 25 degrees C at pH 7.8, whereas the Mn-reconstituted enzyme showed no inactivation after 80 min. A concomitant increase in absorbance at 320 nm was observed with inactivation of the Fe-reconstituted enzyme. Amino acid analysis of the inactivated Fe-reconstituted enzyme showed a decrease of about 0.7 residues of tryptophan/subunit, a value similar to the iron content of the iron-reconstituted enzyme. Three major peptides of the digests of the purified SOD with lysylendopeptidase were separated by a reverse-phase HPLC monitoring at 280 nm. One of the peptides, corresponding to the residues from Gly149 to Lys176, decreased in the HPLC eluent of the H2O2-inactivated SOD to 20% of the amount measured for native SOD. Since this peptide contains only one tryptophan residue, it was concluded that the decomposed tryptophan residue is Trp159, which is located midway between the third and fourth metal ligands, Asp157 and His161, and is conserved in aligned amino acid sequences of all known Fe-SODs and Mn-SODs. Based on these results, we propose that the differences in hydrogen peroxide sensitivities observed for the Fe-SODs and Mn-SODs may be caused by the difference in the identity of the active site metal in the Fe-SODs and Mn-SODs and a tuning of the properties of the iron center in the Fe-SODs.

pH-dependent activity change of superoxide dismutase from Mycobacterium smegmatis.

Superoxide dismutase (SOD), purified from Mycobacterium smegmatis, was found to contain both manganese and iron. Since the Fe and Mn-reconstituted enzymes had specific activities of 190 and 2810 units/mg protein/g atom of metal/mol of subunit, respectively, the Mycobacterial SOD can be classified with SODs showing activity with either iron or manganese as the active-site metal (a cambialistic SOD). Mn-reconstituted enzyme showed an enzymatic reaction rate constant of 1.4 x 10(8) M-1 s-1 at pH 7.8. This rate only slightly increased with decreasing pH. Fe-reconstituted enzyme showed a rate constant of 2.7 x 10(7) M-1 s-1 at pH 7.8, but this rate increased with decreasing pH to become 1.7 x 10(8) M-1 s-1 at pH 5.7 with two pK values of 6.6 and 9.0. These results show that the metal specificity of the enzymatic activity of M. smegmatis superoxide dismutase shows manganese predominance at pH 7.8, but changes to be equal for either metal at acidic pH.

The pH-dependent changes of the enzymic activity and spectroscopic properties of iron-substituted manganese superoxide dismutase. A study on the metal-specific activity of Mn-containing superoxide dismutase.

Manganese-containing superoxide dismutases (Mn-SODs) and iron-containing superoxide dismutases (Fe-SODs) from aerobic bacteria often show high metal specificity for their enzymic activities by a standard assay system using xanthine-xanthine oxidase and cytochrome c. In this study, we have attempted to characterize the structural basis of the metal specificity of manganese-containing SOD (Mn-SOD) using Fe-substituted Mn-SOD prepared from apo-Mn-SOD from Serratia marcescens. The Fe3+ content of the Fe-substituted enzyme was 1.71 +/- 0.14 mol/mol dimer and the specific activity was 34.8 +/- 4.8 units.mg protein-1.mol Fe3+(-1).mol subunit-1. Fe-substituted Mn-SOD was found to react with the superoxide anion at pH 8.1 with a second-order rate constant of 6 x 10(6) M-1 s-1, which is approximately 1% of that of native Mn-SOD at the same pH. However, the rate constant increased with decreasing pH to approximately 10% (5 x 10(7) M-1 s-1) that of native Mn-SOD at pH 6.0 with a pK of 7.0. The visible absorption spectrum and EPR spectrum of Fe-substituted Mn-SOD also showed pH-dependent changes with pK values of 6.6 and 7.2, respectively. Similarly, the affinity of the azide ion, an analog of the superoxide ion, for iron of Fe-substituted Mn-SOD increased with decreasing pH, with a pK value of 7.0 (e.g. Kd = 0.1 mM at pH 6.2 and 0.9 mM at pH 8.2). The similarity of these pK values suggests that the activity, the spectral changes and the affinity of the azide ion for iron are derived from the same change in the metal environment. After comparison with the reported pK values (around 9) of similar pH-dependent changes in the spectra, the enzymic activity and the affinity of azide for iron of Fe-SOD from Escherichia coli, we proposed that the difference in the pK values of a hydroxide ion binding to iron between Fe-substituted Mn-SOD and Fe-SOD may cause the different pH dependencies of these changes in each SOD.

[Serum superoxide dismutase (SOD) activity in patients with renal disease by a spintrap method using electron spin resonance (ESR)].

Superoxide dismutase (SOD) activity in serum samples of patients with chronic glomerulonephritis (CGN) and chronic renal failure (CRF) was measured by a spin trap method using electron spin resonance (ESR). Twenty-three patients with CGN, 10 patients with CRF and 10 healthy adults were examined. Among 23 patients with CGN, there were 12 patients with IgA nephropathy and one patient with membranous nephropathy diagnosed by immunofluorescence of renal biopsy specimens. Other CGN patients were diagnosed by its clinical criteria. The serum activity of SOD in patients with CGN or CRF was significantly higher than those in healthy adults (p < 0.05). The serum SOD activity in patients with CRF was also higher than those in patients with CGN (p < 0.05). Marked high levels of serum SOD activity were observed histologically in the advanced stage of IgA nephropathy. These results suggest an increase in serum SOD activity may reflect renal injuries in patients with CGN and CRF.

Iron- and manganese-containing superoxide dismutases from Methylomonas J: identity of the protein moiety and amino acid sequence.

Mn-superoxide dismutase (SOD) and Fe-SOD were isolated from Methylomonas J, an aerobic methylotrophic bacterium, grown in methylamine media containing either manganese (Mn-rich medium) or iron (Fe-rich medium), respectively. The specific activity of the Mn-SOD was 2250 units mg-1 (mol of Mn)-1 (mol of dimer)-1, and the metal content of the enzyme was 0.98 mol of Mn and 0.12 mol of Fe per mole of dimer, while those of Fe-SOD were 88.5 units mg-1 (mol of Fe)-1 (mol of dimer)-1 and 1.04 mol of Fe and 0.02 mol of Mn. The electrophoretic mobilities in the presence of sodium dodecyl sulfate, with or without urea, and the chromatographic behavior on an HPLC column using an octadodecyl silicated column and a gel permeation column were identical. Amino acid compositions were practically indistinguishable in both SODs. The enzyme activity was restored by dialysis of an apoprotein obtained from the Mn-enzyme with either manganese sulfate or ferrous ammonium sulfate up to an activity level similar to that for the native Mn-SOD and the native Fe-SOD, respectively. The same result has been reported with the reconstitution using an apoprotein obtained from the Fe-enzyme [Yamakura, F., Matsumoto, T., & Terauchi, K. (1990) Free Radical Res. Commun. (in press)]. These results suggest the possibility that both types of SODs are composed of a single apoprotein synthesized in cells grown in either the Fe-rich medium or the Mn-rich medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of superoxide dismutases in Photobacterium leiognathi.

We investigated the induction of Cu,Zn-SOD (bacteriocuprein) and Fe-SOD in Photobacterium leiognathi DK-A1 which was isolated from the light organ of the squid, Droteuthis kensaki. The induction of superoxide dismutases depended on the addition of paraquat to the medium. Induction of SOD by paraquat was attributed mostly to the bacteriocuprein by measuring of the activities of both SODs by using densitometry of isoelectrofocusing gel. When paraquat was added to the culture at various times in the early log phase of growth, the most efficient induction of the SODs, which was measured at the time of harvesting the cells (17 hours after inoculation), was observed when paraquat was added at 60 min after the inoculation. Catalase was not significantly induced by the addition of paraquat or increasing of oxygen concentration. We developed an assay of SOD by modification of a cytochrome c-xanthine oxidase method using a computer equipped absorption spectrophotometer.

Isolation of Mn-SOD and low active Fe-SOD from Methylomonas J; consisting of identical proteins.

Cultures of Methylomonas J, an aerobic methylotrophic bacterium, were grown both in Mn-rich and Fe-rich media. Crude extracts of the cultures from the Mn-rich and Fe-rich medium showed a specific activity of 12.2 and 0.6 units/mg by a cytochrome c-xanthine oxidase method and 19.4 and 1.3 units/mg by an ESR method, respectively. We isolated Mn-SOD and Fe-SOD from the bacteria grown in the Mn-rich and Fe-rich mediums, respectively. Specific activity and metal contents of the Mn-enzyme were 2,250 units/mg/g-atom Mn and Mn = 0.98 and Fe = 0.12 (g-atoms/mol dimer), while those of the Fe-enzyme were 61 units/mg/g-atom Fe and Mn = 0.02 and Fe = 1.08. No difference of physicochemical properties of the Fe- and Mn-enzymes were detected. Furthermore, enzyme activity was restored by dialysis of an apoprotein obtained from the Fe-enzyme with either manganese sulfate or ferrous ammonium sulfate.

Changes in free amino acid concentration in the hemolymph of the female Culex pipiens pallens (Diptera: Culicidae), after a blood meal.

The analysis of the free amino acids in the hemolymph of female Culex pipiens pallens L. indicated that asparagine, glutamine, glycine, leucine, serine, threonine, tyrosine, and valine noticeably increased in concentration during a blood meal digestion at 22 degrees C. The concentrations started to rise at about 4 h, reaching maximal level at 12-24 h, and then gradually returned to the prefeeding level by the fifth day after the blood meal. Before the sharp increase, the concentrations of asparagine, glutamine, glycine, serine, threonine, and tyrosine declined slightly during the first 1-2 h, probably because of hemolymph dilution by water absorbed from the blood meal. Histidine and lysine also showed a minor decrease soon after blood feeding, followed by a moderate but noticeable increase, delaying maximal concentrations until 48 h after the blood meal. Proline and alanine both exhibited a high hemolymph content and changed greatly, but with a large variation between the two samples analyzed. The changing pattern of individual hemolymph amino acids depended to a large extent upon the content of each amino acid in the blood meal given to the mosquitoes. Other free amino acids detected in the hemolymph were at very small concentrations and except for phenylalanine did not show any changes after a blood meal. The total free amino acid concentration was 50 nmol (or 6 micrograms) per microliters hemolymph before a blood meal, and increased to a maximal concentration of 88 nmol (or 11 micrograms) per microliters hemolymph at 18 h after the blood meal.

Purification and some properties of a 2Fe ferredoxin in Pseudomonas ovalis.

A [2Fe-2S] ferredoxin was found in Pseudomonas ovalis which was grown in a medium supplemented with glucose and ammonium sulfate. The molecular weight of the 2Fe ferredoxin was estimated to be 13,000. It contained 2.2 gramatoms of non-heme iron and 2.3 gramatoms of acid-labile sulfur per mole protein. The absorption and circular dichroism spectra were characteristic of those of [2Fe-2S] type ferredoxins, especially adrenodoxin and putidaredoxin. The electron paramagnetic resonance spectrum of the reduced protein showed an axial symmetry (g = 2.020, g = 1.939). The amino acid composition was determined.

Difference between amino acid residues in the metal-ligand environments of iron- and manganese-superoxide dismutases.

Alignment of the amino acid sequences of the Pseudomonas ovalis and Photobacterium leiognathi iron-superoxide dismutases (Fe-SODs) with the known sequences of the manganese-superoxide dismutases (Mn-SODs) shows that both types of SOD are highly homologous (33-53% identity) and share residues for the metal coordination. The amino acid residues that form the environment of the metal ions appear to be also conserved between the Fe- and Mn-SODs, except that the Phe-84 and Gln-154 in the Mn-SODs are replaced by Tyr and Ala, respectively, in the Fe-enzymes. Since this latter residue contributes to formation of the hydrophobic metal-ligand environment through hydrogen bonding with Trp-133 and Tyr-34 in the Mn-SODs, its substitution by Ala should cause different micro environments between the metal centers of the Fe- and Mn-SODs. This difference may account for the metal specificity of both types of SODs demonstrated by previous reconstitution experiments.

Amino acid sequence of iron-superoxide dismutase from Pseudomonas ovalis.

The amino acid sequence of iron-superoxide dismutase from Pseudomonas ovalis was deduced by the analyses of peptides derived from limited hydrolysis of the aminoethylated or pyridylethylated apoprotein with trypsin, Staphylococcus aureus V8 protease, and dilute acid hydrolysis. The polypeptide chain contains 195 amino acid residues and has a calculated Mr of 21,421. The sequence is highly homologous (65% identity) to the recently published sequence of the iron-superoxide dismutase from Photobacterium leiognathi. It is also homologous to the known sequences of the manganese-superoxide dismutase by sharing 33-53% identical residues. Alignment of the superoxide dismutase sequences and the available structural information from X-ray crystallography suggest that the ligands to the iron in the P. ovalis superoxide dismutase are His-26, His-74, Asp-156 and His-160, which align with the ligands to the manganese in the Thermus thermophilus manganese-superoxide dismutase. The sequence information of the P. ovalis dismutase will facilitate refinement of the X-ray crystallographic data that are now available at 2.9 A resolution.

Inactivation of Pseudomonas iron-superoxide dismutase by hydrogen peroxide.

Pseudomonas Fe-superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) is inactivated by hydrogen peroxide by a mechanism which exhibits saturation kinetics. The pseudo-first-order rate constant of the inactivation increased with increasing pH, with an inflection point around pH 8.5. Two parameters of the inactivation were measured in the pH range 7.8 to 9.0; the total H2O2 concentration at which the enzyme is half-saturated (K inact) was found to be independent of pH (30 mM) and the maximum rate constant for inactivation (k max) increased progressively with increasing pH, from 3.3 min-1 at pH 7.8 to 21 min-1 at pH 9.0. This evidence suggests the presence of an ionization group (pKa approximately 8.5) which does not participate in the binding of H2O2 but which affects the maximum inactivation rate of the enzyme. The loss of dismutase activity of the Fe-superoxide dismutase is accompanied by a modification of 1.6, 1.1 and 0.9 residues of tryptophan, histidine and cysteine, respectively. Since the amino acid residues of the Cr-substituted enzyme, which has no enzymatic activity, were not modified by H2O2, the active iron of the enzyme is essential for the modification of the amino acid residues.