Denaturation and renaturation of myeloperoxidase. Consequences for the nature of the prosthetic group.

The effects of the chaotropic agent, guanidine HCl, on the chlorinating activity, optical absorption, EPR, and resonance Raman spectra of myeloperoxidase have been studied. In the presence of the agent the Soret optical absorption of the reduced enzyme (lambda max = 474 nm) is blue shifted to 448 nm, a position similar to heme alpha-containing enzymes. The chlorinating activity of the enzyme disappears, and EPR spectra show a loss of intensity of the rhombic high spin heme signals (gx = 6.9; gy = 5.4) and the appearance of a more axial high spin signal (gx = gy = 6.0). Surprisingly the effects of guanidine HCl are partly reversible. Upon decreasing the concentration of the chaotropic agents by dilution, both the chlorinating activity and the original optical spectrum of native reduced enzyme (lambda max = 474 nm) are partly restored. The resonance Raman spectra of denatured cyanomyeloperoxidase are less complicated than those of native myeloperoxidase, which have been interpreted previously to suggest an iron chlorin chromophore. The multiple lines in the oxidation state marker region are not seen in the spectra of the denatured species. The changes suggest that upon denaturation the macrocycle is converted into a more symmetric structure. Since the effects on the optical absorption spectrum are reversible we speculate that, in the native enzyme, an apparent porphyrin macrocycle undergoes a reversible interaction with amino acid residues in the protein which creates an asymmetry in the electronic distribution of the macrocycle. Comparison of the Raman spectra of denatured cyanomyeloperoxidase with those of analogous heme alpha model complexes suggests the presence of a formyl group in the denatured species; our data, however, demonstrate that the chromophore structure is not identical to heme alpha and may contain a different C beta substitution on the ring macrocycle.     

Type 1, blue copper proteins constitute a respiratory nitrite-reducing system in Pseudomonas aureofaciens

Pseudomonas aureofaciens truncates the respiratory reduction of nitrate (denitrification) at the level of N2O. The nitrite reductase from this organism was purified to apparent electrophoretic homogeneity and found to be a blue copper protein. The enzyme contained 2 atoms of copper/85 kDa, both detectable by electron paramagnetic resonance (EPR) spectroscopy. The protein was dimeric, with subunits of identical size (40 +/- 3 kDa). Its pI was 6.05. The EPR spectrum showed an axial signal g at 2.21(8) and g at 2.04(5). The magnitude of the hyperfine splitting (A parallel = 6.36 mT) indicated the presence of type 1 copper only. The electronic spectrum had maxima at 280 nm, 474 nm and 595 nm (epsilon = 7.0 mM-1 cm-1), and a broad shoulder around 780 nm. A copper protein of low molecular mass (15 kDa), with properties similar to azurin, was also isolated from P. aureofaciens. The electronic spectrum of this protein showed a maximum at 624 nm in the visible range (epsilon = 2.5 mM-1 cm-1) and pronounced structures in the ultraviolet region. The EPR parameters were g parallel = 2.26(6) and g perpendicular = 2.05(6), with A parallel = 5.8 mT. The reduced azurin transferred electrons efficiently to nitrite reductase; the product of nitrite reduction was nitric oxide. The specific nitrite-reducing activity with ascorbate-reduced phenazine methosulfate as electron donor was 1 mumol substrate min-1 mg protein-1. The reaction product again was nitric oxide. Nitrous oxide was the reaction product from hydroxylamine and nitrite and from dithionite-reduced methyl viologen and nitrite. No 'oxidase' activity could be demonstrated for the enzyme. Our data disprove the presumed exclusiveness of cytochrome cd1 as nitrite reductase within the genus Pseudomonas.     

Effect of nitrite on cytochrome oxidase

Nitrite causes changes in the optical and EPR spectra of cytochrome oxidase from heart and alters the spectral, redox and basic properties of cytochrome c. No utilization of nitrite by cytochrome oxidase was observed. However, nitrite inhibits the superoxide dismutase and oxidase activities of the enzyme. Changes in the properties of cytochrome oxidase were observed under effect of some products of nitrite reduction, e. g. nitric oxide, hydroxylamine, hydrazine; nitrate has no effect on the optical and EPR spectra or on the enzyme activity.     

Demonstration that spleen green hemeprotein is identical to granulocyte myeloperoxidase

The biochemical, spectroscopic, enzymatic, and redox properties of spleen myeloperoxidase, a peroxidase formerly called "spleen green hemeprotein," and granulocyte myeloperoxidase were compared by electrophoresis, amino-terminal amino acid sequences, optical and EPR spectroscopy, steady-state enzyme kinetics, and oxidation-reduction potential. The spleen enzyme exhibits properties indistinguishable from those of the granulocyte enzyme. We conclude that the spleen enzyme is indeed identical to granulocyte myeloperoxidase.     

Isolation procedure and some properties of myeloperoxidase from human leucocytes.

1. A rapid isolation procedure with a high yield for pure myeloperoxidase (donor:H2O2 oxidoreductase, EC 1.11.1.7) from normal human leucocytes is described. The enzyme was solubilized from leucocytes with the detergent, cetyltrimethylammonium bromide, and purified to apparent homogeneity. The yield of the enzyme was 17% with an absorbance ratio A430nm/A280nm = 0.85. 2. The purified enzyme showed three isoenzyme bands after polyacrylamide gel electrophoresis; ultracentrifuge studies indicated one homogeneous band with a molecular weight of 144 000. After reduction of myeloperoxidase, sodium dodecyl sulfate gel electrophoresis resolved an intense band (63 000 daltons) and a weak band (81 000 daltons). 3. The carbohydrate content of the enzyme was at least 2.5%. Mannose, glucose and N-acetylglucosamine were present. The amino acid composition is reported. 4. The EPR spectrum exhibited a high-spin heme signal with rhombic symmetry (gx = 6.92, gy = 5.07 and gz = 1.95). Upon acidification this signal was converted into a signal with more axial symmetry (g perpendicular = 5.89). At high pH (9.5) the EPR spectrum of the enzyme only shows low-spin ferric heme resonances. The circular dichroism spectra of ferric and ferrous myeloperoxidase in the visible and ultraviolet region show maxima and minima in ellipticity.     

Some properties of human eosinophil peroxidase, a comparison with other peroxidases

Eosinophil peroxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) was isolated from outdated human white blood cells. The purified enzyme has a molecular weight of 71000 +/- 1000. The enzyme is composed of two subunits, of Mr 58000 and 14000, in a 1:1 stoichiometry. Amino-acid analyses showed that eosinophil peroxidase has a high content of the amino acids arginine, leucine and aspartic acid. The millimolar absorbance coefficient of the Soret band at 412 nm of eosinophil peroxidase was determined. Three independent methods yield a value for epsilon 412nm of 110 +/- 4 mm-1 X cm-1. Purified eosinophil peroxidase showed a homogeneous high-spin EPR signal with rhombic symmetry (gx = 6.50; gy = 5.40; gz = 1.982) for the haem group. EPR spectroscopy of low-spin cyanide and azide derivatives of eosinophil peroxidase, lactoperoxidase, myeloperoxidase and catalase revealed that the haem-ligand structure of eosinophil peroxidase is closely related to lactoperoxidase, whereas that of myeloperoxidase shows great resemblance to catalase.     

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.     

The effect of chloride on the redox and EPR properties of myeloperoxidase

Myeloperoxidase was purified from human polymorphonuclear leukocytes and the effect of chloride upon the EPR and potentiometric properties was studied. The redox titration between the ferrous and ferric states of the enzyme yielded n = 1 Nernst plots between pH 9 and 4, with clear isosbestic points in the optical spectra during the redox change. The midpoint potential (Em) between the ferric and ferrous forms of the enzyme exhibited a pH-dependent change between pH 4 and 9, and the effect of added chloride ion indicated that Cl- competed with OH- for a binding site on the enzyme. Interestingly, the pH dependence of the Em indicated that the overall redox reactions of the enzyme was: ferric myeloperoxidase + 2e- + 1H+ = ferrous myeloperoxidase. Myeloperoxidase exhibited a rhombic high spin EPR signal which exhibited reduced rhombicity upon the binding of chloride. Our results strongly suggest that chloride binds to the sixth coordination position of the chlorin iron in myeloperoxidase by replacing the water which is the sixth ligand in the resting state. It is also concluded that the two iron centers are identical and that there is no interaction between them.     

On the purification of nitrite reductase from Thiobacillus denitrificans and its reaction with nitrite under reducing conditions.

Nitrite reductase (cytochrome $\text{cd}$) from $\text{T. denitrificans}$ has been crystallized in high yield in three simple and rapid steps. The spectral absorption ratio at 408 to 280 nm was 1.52. Light absorption spectra in the oxidized and reduced states were virtually identical to those of nitrite reductase from $\text{P. aeruginosa}$. EPR spectroscopy of nitrite reductase at 12° showed a low-spin ferric heme resonance with g-values at 2.52, 2.45 and 1.73 assigned to the d-heme. Reaction of nitrite reductase with nitrite in the presence of the reducing systems [(ascorbate + PMS) or sulfide] resulted in the formation of nitric oxide (confirmed by gas chromatography) which reacted with both $\text{c}$- and $\text{d}$-hemes of nitrite reductase yielding an EPR-detectable enzyme-NO complex with g-values at 2.07, 2.04 and 1.99 and a ¹⁴N hyperfine splitting constant of 22.5 gauss. The amount of nitric oxide produced enzymatically with sulfide as electron donor was only 5% of that found when ascorbate plus PMS served as reductant.To our knowledge the detection of the unique enzyme-NO complex is the first definitive EPR evidence for the mandatory liganding of nitric oxide with pure nitrite reductase during nitrite reduction.     

Characterization of a novel copper-haem c dissimilatory nitrite reductase from Ralstonia pickettii

NiRs (nitrite reductases) convert nitrite into NO in the denitrification process. RpNiR (Ralstonia pickettii NiR), a new type of dissimilatory Cu-containing NiR with a C-terminal haem c domain from R. pickettii, has been cloned, overexpressed in Escherichia coli and purified to homogeneity. The enzyme has a subunit molecular mass of 50515 Da, consistent with sequence data showing homology to the well-studied two-domain Cu NiRs, but with an attached C-terminal haem c domain. Gel filtration and combined SEC (size-exclusion chromatography)-SAXS (small angle X-ray scattering) analysis shows the protein to be trimeric. The metal content of RpNiR is consistent with each monomer having a single haem c group and the two Cu sites being metallated by Cu(2+) ions. The absorption spectrum of the oxidized as-isolated recombinant enzyme is dominated by the haem c. X-band EPR spectra have clear features arising from both type 1 Cu and type 2 Cu centres in addition to those of low-spin ferric haem. The requirements for activity and low apparent K(m) for nitrite are similar to other CuNiRs (Cu-centre NiRs). However, EPR and direct binding measurements of nitrite show that oxidized RpNiR binds nitrite very weakly, suggesting that substrate binds to the reduced type 2 Cu site during turnover. Analysis of SEC-SAXS data suggests that the haem c domains in RpNiR form extensions into the solvent, conferring a high degree of conformational flexibility in solution. SAXS data yield R(g) (gyration radius) and D(max) (maximum particle diameter) values of 43.4 ? (1 ?=0.1 nm) and 154 ? compared with 28 ? and 80 ? found for the two-domain CuNiR of Alcaligenes xylosoxidans.     

An EPR study of myeloperoxidase in human granulocytes.

1. EPR spectra of human granulocytes (4 - 10(8) cells per ml) show an intense high-spin ferric heme signal with rhombic symmetry (gx = 6.90 and gy = 5.07) for the heme group. These g-values are identical to those of partially purified myeloperoxidase and thus the signal is derived from ferric myeloperoxidase. In chicken granulocytes, which contain little or no myeloperoxidase, only an axial type of heme iron signal, weak in intensity, can be detected at g = 6.0. 2. Upon phagocytosis of latex particles by human granulocytes the high-spin heme signal with rhombic symmetry is slowly converted into a signal with axial symmetry (gx = gy = 6.0), showing that the EPR signals of myeloperoxidase in the intact cell can be used to study the involvement of the enzyme in metabolic changes during phagocytosis.     

Peroxide interaction with pulsed cytochrome oxidase. Optical and EPR studies

EPR and optical analysis of the 420 nm form of cytochrome oxidase (Kumar, C., Naqui, A., and Chance, B. (1984) J. Biol. Chem. 259, 2073-2076) shows that 1) the 420 nm form possesses a 605 nm band, g = 5 EPR signals, and a slightly blue shifted 655 nm band; 2) the reaction of H2O2 with the 420 nm form generates the peroxide complex (Soret band at 427 nm) with the formation of a 580 nm band and abolition of both the 655 nm band and the g = 5 EPR signal. Comparison of our results with past data shows that various forms of oxidase formed from the resting oxidase through different protocols may be identified to be either the 420 nm or the 427 nm form and leads to identification of a peroxy intermediate during oxidase turnover.     

Studies on the origin of the near-infrared (800-900 nm) absorption of cytochrome c oxidase

Data are presented which were collected in the course of the past ten years and bear on the correlation of absorbance at 800 nm and the EPR signal at g = 2 ('copper signal') of cytochrome c oxidase in various states of oxidation and ligation. Both EPR and optical reflectance spectra were obtained at low temperature (-170 to -190 degrees C). For some sets of samples spectra were recorded in the range 500-1100 nm. A particular efFort was made to study this correlation with what are called 'mixed valence' states (Greenwood, C., Wilson, M.T. and Brunori, M. (1974) Biochem. J. 137, 205-215), when cytochrome a and the EPR-detectable copper are thought to be oxidized and the other components reduced and vice versa. These data show no evidence that the copper component of cytochrome oxidase which has so far not been detected by EPR makes a contribution to the absorption between 800 and 900 nm exceeding 10-15% of the total, which is close to or within the error of the respective measurements. For the various states of the oxidase examined in this work the 700-800 nm region did not appear to be more useful than the 800-900 nm region for determining the state of the EPR-undetectable copper in a reliable way. These conclusions are in agreement with results presented previously from other laboratories concerning the relationship of optical (approx. 800 nm) and EPR spectroscopic (g = 2) data obtained with the enzyme.     

The role of an iron-sulfur center and siroheme in spinach nitrite reductase.

Purified spinach nitrite reductase, a protein that contains siroheme, is characterized by absorption maxima in the visible region at 385 and 573 nm. On addition of the substrate nitrite, the bands shift to 360 and 570 nm. Dithionite also causes shifts in the maxima of the visible absorption region. Electron paramagnetic resonance studies show that the untreated enzyme contains a high-spin Fe³⁺ heme and that the addition of cyanide, an inhibitor that is competitive with nitrite, results in a spin-state change of the heme. Electron paramagnetic resonance analysis of the enzyme in the presence of dithionite or dithionite plus cyanide indicates the presence of a reduced iron-sulfur center with rhombic symmetry (g-values of 2.03, 1.94, and 1.91). In contrast, when the enzyme is treated with dithionite plus nitrite, the EPR spectrum of an NO-heme complex (g-values of 2.07 and 2.00) is observed. The presence of an iron-sulfur center has also been confirmed by chemical analyses of the nonheme iron and acid-labile sulfide in nitrite reductase. These results are discussed in terms of a mechanism for nitrite reduction that involves electron transfer between the iron-sulfur center and siroheme.     

Binding of nitrosamines to cytochrome P-450 of liver microsomes.

The interactions of 5 carcinogenic and 1 non-carcinogenic nitrosamines with hepatic microsomal cytochrome (cyt.) P-450 were investigated, using both optical difference and electron paramagnetic resonance (EPR) spectroscopic methods. Liver microsomes from phenobarbital (PB)-pretreated mice and 3-methylcholanthrene (3-MC)-pretreated rats were used, in order to have an increased specific content of cyt. P-450 and cyt. P-448 respectively. The optical and EPR spectral data obtained in the oxidised state suggest that nitrosamines are able to bind both as substrates and as ligands to the hemoprotein cyt. P-450, depending on the concentration of nitrosamine, its chemical identity and the cytochrome species present. After reduction with dithionite or NADPH in the optical difference spectrum a Soret band developed between 444 and 453 nm to an extent, which is dependent on the particular nitrosamine present. This initial nitrosamine-induced spectrum might represent a ferrous nitric oxide (NO)-cyt. P-450 complex. It appears unstable and is converted kinetically into a spectrum lacking a Soret band, but with a predominant absorbance minimum at about 425 nm. A visible band is located at 585 nm. In the EPR spectrum a sharp 3-line signal around g = 2.01 appears concomitantly. Both spectral parameters are typical of a NO-cyt. P-420 complex. These results, in conjunction with metabolic studies, indicate that nitrosamines are denitrosated by a reductive process in which cyt. P-450 appears to be involved. The resulting NO-cyt. P-450 complex denatures to a NO-cyt. P-420 complex when the dioxygen level is not sufficiently high to complete successfully.     

Nitrite oxidoreductase from Nitrobacter hamburgensis: redox centers and their catalytic role

Nitrite oxidoreductase was isolated from mixotrophically grown cells of Nitrobacter hamburgensis. The enzyme purified from heat treated membranes was homogeneous by the criteria of polyacrylamide gel electrophoresis and size exclusion chromatography. The monomeric form consisted of two subunits with M_r 115000 and 65000, respectively. The dimeric form of the enzyme contained 0.70 molybdenum, 23.0 iron, 1.76 zinc, and 0.89 copper. The catalytically active enzyme was investigated by visible and electron paramagnetic resonance spectroscopy (EPR) under oxidizing (as isolated), reducing (dithionite), and turnover (nitrite) conditions. As isolated the enzyme exhibited a complex set of EPR signals between 5–75 K, originating from several ironsulfur and molybdenum (V) centers. Addition of the substrate nitrite, or the reducing agent dithionite resulted in a set of new resonances. The molybdenum and the iron-sulfur centers of nitrite oxidoreductase from Nitrobacter hamburgensis were involved in the transformation of nitrite to nitrate.Abbreviations EPR electron paramagnetic resonance - ICP-AES inductively coupled plasma-atomic emission spectrometry - NaP_i sodium phosphate - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Studies on the origin of the near-infrared (800–900 nm) absorption of cytochrome c oxidase

Data are presented which were collected in the course of the past ten years and bear on the correlation of absorbance at 800 nm and the EPR signal at g = 2 (‘copper signal’) of cytochrome c oxidase in various states of oxidation and ligation. Both EPR and optical reflectance spectra were obtained at low temperature (?170 to ?190°C). For some sets of samples spectra were recorded in the range 500–1100 nm. A particular effort was made to study this correlation with what are called ‘mixed valence’ states (Greenwood, C., Wilson, M.T. and Brunori, M. (1974) Biochem. J. 137, 205–215), when cytochrome a and the EPR-detectable copper are thought to be oxidized and the other components reduced and vice versa. These data show no evidence that the copper component of cytochrome oxidase which has so far not been detected by EPR makes a contribution to the absorption between 800 and 900 nm exceeding 10–15% of the total, which is close to or within the error of the respective measurements. For the various states of the oxidase examined in this work the 700–800 nm region did not appear to be more useful than the 800–900 nm region for determining the state of the EPR-undetectable copper in a reliable way. These conclusions are in agreement with results presented previously from other laboratories concerning the relationship of optical (approx. 800 nm) and EPR spectroscopic (g = 2) data obtained with the enzyme.     

Effect of acetaminophen on the myeloperoxidase-hydrogen peroxide-nitrite mediated oxidation of LDL

Myeloperoxidase, in the presence of hydrogen peroxide and nitrite, promotes the lipid peroxidation of low density lipoprotein (LDL); the modified lipoprotein is then capable of being readily endocytosed by macrophages. Since acetaminophen has been shown to inhibit the leukocyte myeloperoxidase antimicrobial system and is, under certain experimental conditions, an antioxidant, the effect of acetaminophen on the myeloperoxidase-hydrogen peroxide-nitrite mediated oxidation of LDL was examined. The content of LDL lipid hydroperoxides after incubation with 50 nM myeloperoxidase, 100 microM nitrite and a hydrogen peroxide generating system for 6 h was reduced by approx. 80% in the presence of 25-250 microM acetaminophen. The production of thiobarbituric acid-reactive substances was also inhibited by acetaminophen to a similar extent. Acetylsalicylic acid (25-100 microM) did not inhibit LDL lipid peroxidation mediated by the myeloperoxidase enzyme system. LDL, treated with myeloperoxidase, hydrogen peroxide and nitrite for 14 h, was metabolized by macrophages to a much greater extent than native LDL. The presence of acetaminophen prevented the modification of LDL; the lipoprotein was metabolized by macrophages to the same extent as was native LDL. These results demonstrate that acetaminophen is a potent inhibitor of the myeloperoxidase-hydrogen peroxide-nitrite mediated modification of LDL.     

EPR and optical changes of the photosystem II reaction center produced by low temperature illumination

Electron paramagnetic resonance (EPR) and absorption spectroscopy have been used to study the low temperature photochemical behavior of the Photosystem II D-1/D-2/ cytochrome b559 reaction center complex. The reaction center displays large triplet state EPR signals which are attenuated after actinic illumination at low temperatures in the presence of sodium dithionite. Concomitant with the triplet attenuation is the buildup of a structured radical signal with an effective g value of 2.0046 and a peak-to-peak width of 11.9 G. The structure in the signal is suggestive of it being comprised in part of the anion radical of pheophytin a. This assignment is corroborated by low temperature optical absorbance measurements carried out after actinic illumination at the low temperatures which show absorption bleachings at 681 nm, 544 nm and 422 nm and an absorbance buildup at 446 nm indicating the formation of reduced pheophytin.Abbreviations EPR electron paramagnetic resonance     

Heme proteins mediate the conversion of nitrite to nitric oxide in the vascular wall

Nitric oxide (NO) has been shown to be the endothelium-derived relaxing factor (EDRF), and its impairment contributes to a variety of cardiovascular disorders. Recently, it has been recognized that nitrite can be an important source of NO; however, questions remain regarding the activity and mechanisms of nitrite bioactivation in vessels and its physiological importance. Therefore, we investigated the effects of nitrite on in vivo hemodynamics in rats and in vitro vasorelaxation in isolated rat aorta under aerobic conditions. Studies were performed to determine the mechanisms by which nitrite is converted to NO. In anesthetized rats, nitrite dose dependently decreased both systolic and diastolic blood pressure with a threshold dose of 10 microM. Similarly, nitrite (10 microM-2 mM) caused vasorelaxation of aortic rings, and NO was shown to be the intermediate factor responsible for this activity. With the use of electrochemical as well as electron paramagnetic resonance (EPR) spectroscopy techniques NO generation was measured from isolated aortic vessels following nitrite treatment. Reduction of nitrite to NO was blocked by heating the vessel, suggesting that an enzymatic process is involved. Organ chamber experiments demonstrated that aortic relaxation induced by nitrite could be blocked by both hemoglobin and soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ). In addition, both electrochemical and EPR spin-trapping measurements showed that ODQ inhibits nitrite-mediated NO production. These findings thus suggest that nitrite can be a precursor of EDRF and that sGC or other heme proteins inhibited by ODQ catalyze the reduction of nitrite to NO.