The iron-sulfur cluster composition of Escherichia coli nitrate reductase

Nitrate reductase from Escherichia coli has been investigated by low-temperature magnetic circular dichroism and electron paramagnetic resonance (EPR) spectroscopies, as well as by Fe-S core extrusion, to determine the Fe-S cluster composition. The results indicate approximately one 3Fe and three or four [4Fe-4S]2+,1+ centers/molecule of isolated enzyme. The magnetic circular dichroism spectra and magnetization characteristics show the oxidized and reduced 3Fe and [4Fe-4S] centers to be electronically analogous to those in bacterial ferredoxins. The form and spin quantitation of the EPR spectra from [4Fe-4S]1+ centers in the reduced enzyme were found to vary with the conditions of reduction. For the fully reduced enzyme, the EPR spectrum accounted for between 2.9 and 3.5 spins/molecule, and comparison with partially reduced spectra indicates weak intercluster magnetic interactions between reduced paramagnetic centers. In common with other Fe-S proteins, the 3Fe center was not extruded intact under standard conditions. The results suggest that nitrate reductase is the first example of a metalloenzyme where enzymatic activity is associated with a form that contains an oxidized 3Fe center. However, experiments to determine whether or not the 3Fe center is present in vivo were inconclusive.     

L-SOD发挥活性作用的可能机制

氧化剂、还原剂处理前后,Ｌ－ＳＯＤ的活性及紫外光谱发生变化,Ｈ２Ｏ２使Ｆｅ（Ⅲ）吸收增强,同时钝化Ｌ－ＳＯＤ的活性;加入保险粉后,Ｌ－ＳＯＤ重新活化,Ｆｅ（Ⅲ）吸收减弱．ＮＥＭ封闭Ｃｙｓ后,Ｌ－ＳＯＤ紫外吸收谱发生变化,且活性减弱．说明Ｆｅ辅基及Ｃｙｓ是活性发挥的必需基团．     

Interactions of microsomal cytochrome P-450 with spin-labeled substrate analogs

The iodine-containing stable iminoxyl radicals with various distances between the N-O-group and the iodine atom are proposed to be used to study the structure of the active center of the microsomal cytochrome P-450. The radicals used induce changes in the optical spectra of the Fe3+ ion located in the active center of the enzyme, as in the case of type 1 substrates and inhibit essentially the microsomal oxidation of cytochrome P-450 substrates of type 1 and 2. This inhibition is neither due to suppression of the NADPH-cytochrome c reductase activity nor to cytochrome P-450 conversion to cytochrome P-420. Cytochrome P-450 substrates (aminopyrine) protect the enzyme against the radical-induced inactivation. The iodine-containing radicals are covalently bound to cytochrome P-450 in the vicinity of active center. The values of dissociation constants for the reversible enzyme-radical constants and the rate constants for the monomolecular transformation in the complex, k, were determined. The EPR method was used to detect the coupling between Fe3+ and the radical located in the active center of cytochrome P-450. The saturation curves of radical SPR spectra at 77 degrees K were employed to determine the contribution of Fe3+ to the relaxation time, T1, of the radicals covalently bound to cytochrome P-450 and to estimate the distances between the Fe3+ ion and the N-O-group of these radicals in the enzyme active center.     

Low-spin sulfite reductases: a new homologous group of non-heme iron-siroheme proteins in anaerobic bacteria

Two new low molecular weight proteins with sulfite reductase activity, isolated from Methanosarcina barkeri (DSM 800) and Desulfuromonas acetoxidans (strain 5071), were studied by EPR and optical spectroscopic techniques. Both proteins have visible spectra similar to that of the low-spin sulfite reductase of Desulfovibrio vulgaris strain Hildenborough and no band at 715 nm, characteristic of high-spin Fe3+ complexes in isobacteriochlorins is observed. EPR shows that as isolated the siroheme is in a low-spin ferric state (S = 1/2) with g-values at 2.40, 2.30 and 1.88 for the Methanosarcina barkeri enzyme and g-values at 2.44, 2.33 and 1.81 for the Desulfuromonas acetoxidans enzyme. Chemical analysis shows that both proteins contain one siroheme and one [Fe4S4] center per polypeptidic chain. These results suggest that the low molecular weight, low-spin non-heme iron siroheme proteins represent a new homologous class of sulfite reductases common to anaerobic microorganisms.     

Mutational and spectroscopic studies of the significance of the active site glutamine to metal ion specificity in superoxide dismutase

We are addressing the puzzling metal ion specificity of Fe- and Mn-containing superoxide dismutases (SODs) [see C.K.Vance, A.-F. Miller, J. Am. Chem. Soc. 120(3) (1998) 461–467]. Here, we test the significance to activity and active site integrity of the Gln side chain at the center of the active site hydrogen bond network. We have generated a mutant of MnSOD with the active site Gln in the location characteristic of Fe-specific SODs. The active site is similar to that of MnSOD when Mn²⁺, Fe³⁺ or Fe²⁺ are bound, based on EPR and NMR spectroscopy. However, the mutant’s Fe-supported activity is at least 7% that of FeSOD, in contrast to Fe(Mn)SOD, which has 0% of FeSOD’s activity. Thus, moving the active site Gln converts Mn-specific SOD into a cambialistic SOD and the Gln proves to be important but not the sole determinant of metal-ion specificity. Indeed, subtle differences in the spectra of Mn²⁺, Fe³⁺ and ¹H in the presence of Fe²⁺ distinguish the G77Q, Q146A mut-(Mn)SOD from WT (Mn)SOD, and may prove to be correlated with metal ion activity. We have directly observed the side chain of the active site Gln in Fe²⁺SOD and Fe²⁺(Mn)SOD by ¹⁵N NMR. The very different chemical shifts indicate that the active site Gln interacts differently with Fe²⁺ in the two proteins. Since a shorter distance from Gln to Fe and stronger interaction with Fe correlate with a lower E_m in Fe(Mn)SOD, Gln has the effect of destabilizing additional electron density on the metal ion. It may do this by stabilizing OH⁻ coordinated to the metal ion.     

Active site structure of SoxB-type cytochrome bo3 oxidase from thermophilic Bacillus

Two-subunit SoxB-type cytochrome c oxidase in Bacillus stearothermophilus was over-produced, purified, and examined for its active site structures by electron paramagnetic resonance (EPR) and resonance Raman (RR) spectroscopies. This is cytochrome bo3 oxidase containing heme B at the low-spin heme site and heme O at the high-spin heme site of the binuclear center. EPR spectra of the enzyme in the oxidized form indicated that structures of the high-spin heme O and the low-spin heme B were similar to those of SoxM-type oxidases based on the signals at g=6.1, and g=3.04. However, the EPR signals from the CuA center and the integer spin system at the binuclear center showed slight differences. RR spectra of the oxidized form showed that heme O was in a 6-coordinated high-spin (nu3 = 1472 cm(-1)), and heme B was in a 6-coordinated low-spin (nu3 = 1500 cm(-1)) state. The Fe2+-His stretching mode was observed at 211 cm(-1), indicating that the Fe2+-His bond strength is not so much different from those of SoxM-type oxidases. On the contrary, both the Fe2+-CO stretching and Fe2+-C-O bending modes differed distinctly from those of SoxM-type enzymes, suggesting some differences in the coordination geometry and the protein structure in the proximity of bound CO in cytochrome bo3 from those of SoxM-type enzymes.     

Evidence for siroheme-Fe4S4 interaction in spinach ferredoxin-sulfite reductase

Spinach ferredoxin-sulfite reductase (SiR) contains one siroheme and one Fe4S4 center per polypeptide subunit. The heme is entirely in the high-spin Fe3+ state in the oxidized enzyme. When SiR is photochemically reduced with ethylenediaminetetraacetate (EDTA)-deazaflavin, the free enzyme and its CN- and CO complexes show changes in absorption spectra associated with the heme even after the heme has been reduced from the Fe3+ to the Fe2+ state. With CO- or CN--SiR, these spectral changes are associated with the appearance of a classical "g = 1.94" type of EPR spectrum characteristic of reduced Fe4S4 centers. The line shapes and exact g values of the g = 1.94 EPR spectra vary with the nature of the ligand bound to the heme Fe. Photoreduction of free SiR results in production of a novel type of EPR signal, with g = 2.48, 2.34, and 2.08 in the fully reduced enzyme; this signal accounts for 0.6 spin per heme. (A small g = 1.94 type EPR signal, representing 0.2 spin per heme, is also found.) These data suggest the presence of a strong magnetic interaction between the siroheme and Fe4S4 centers in spinach SiR, this interaction giving rise to different EPR signals depending on the spin state of the heme Fe in the reduced enzyme.     

超氧化物歧化酶活性中心中铜离子的氧化还原研究

用电子顺磁共振EPR技术研究铜锌超氧化物歧化酶(Cu·Zn-SOD)与底物(O_2~(·-)反应达到平衡态时铜离子的EPR波谱表明,在平衡态时的铜离子处于还原态。用还原剂H_2O_2、NaBH_4处理Cu·Zn-SOD后,酶活力变化不同,电泳行为也不同。用NaBH_4处理SOD其活性及电泳行为接近天然酶,但经H_2O_2还原后的酶活性损失严重,电泳后出现多条色带。     

Kinetic and spectroscopic studies on the quercetin 2,3-dioxygenase from Bacillus subtilis

Quercetin 2,3-dioxygenase from Bacillus subtilis (QueD) converts the flavonol quercetin and molecular oxygen to 2-protocatechuoylphloroglucinolcarboxylic acid and carbon monoxide. QueD, the only known quercetin 2,3-dioxygenase from a prokaryotic organism, has been described as an Fe2+-dependent bicupin dioxygenase. Metal-substituted QueDs were generated by expressing the enzyme in Escherichia coli grown on minimal media in the presence of a number of divalent metals. The addition of Mn2+, Co2+, and Cu2+ generated active enzymes, but the addition of Zn2+, Fe2+, and Cd2+ did not increase quercetinase activity to any significant level over a control in which no divalent ions were added to the media. The Mn2+- and Co2+-containing QueDs were purified, characterized by metal analysis and EPR spectroscopy, and studied by steady-state kinetics. Mn2+ was found to be incorporated nearly stoichiometrically to the two cupin motifs. The hyperfine coupling constant of the g = 2 signal in the EPR spectra of the Mn2+-containing enzyme showed that the two Mn2+ ions are ligated in an octahedral coordination. The turnover number of this enzyme was found to be in the order of 25 s(-1), nearly 40-fold higher than that of the Fe2+-containing enzyme and similar in magnitude to that of the Cu2+-containing quercertin 2,3-dioxygenase from Aspergillus japonicus. In addition, kinetic and spectroscopic data suggest that the catalytic mechanism of QueD is different from that of the Aspergillus quercetinases but similar to that proposed for the extradiol catechol dioxygenases. This study provides evidence that Mn2+ might be the preferred cofactor for this enzyme and identifies QueD as a new member of the manganese dioxygenase family.     

牛红细胞超氧化物歧化酶活性中心的紫外光谱研究

应用差示分光光度法研究了牛红细胞Ｃｕ２Ｚｎ２ＳＯＤ的紫外光谱,归属和讨论了酶活性中心金属离子与配体间全部电荷转移谱带,给出了相应的配体轨道光学电负性,特别研究了涉及Ｚｎ２＋的电荷转移谱带．     

Nitrogenase. VIII. M?ssbauer and EPR spectroscopy. The MoFe protein component from Azotobacter vinelandii OP.

We have studied the molybdenum-iron protein (MoFe protein, also known as component I) from Azobacter vinelandi using M?ssbauer spectroscopy and electron paramagnetic resonance on samples enriched with 57Fe. These spectra can be interpreted in terms of two EPR active centers, each of which is reducible by one electron. A total of four different chemical environments of Fe can be discerned. One of them is a cluster of Fe atoms with a net electronic spin of 3/2, one of them is high-spin ferrous iron and the remaining two are iron in a reduced state (probably in clusters). The results are as follows: Chemical analysis yields 11.5 Fe atoms and 12.5 labile sulfur atoms per molybdenum atom; the molecule contains two Mo atoms per 300 000 daltons. The EPR spectrum of the MoFe protein exhibits g values at 4.32, 3.65 and 2.01, associated with the ground state doublet of a S = 3/2 spin system. The spin Hamiltonian H = D(S2/z minus 5/4 + lambda(S2/x minus S2/y)) + gbeta/o S-H fits the experimental data for go = 2.00 and lambda = 0.055. Quantitative analysis of the temperature dependence of the EPR spectrum yields D/k = 7.5 degrees K and 0.91 spins/molybdenum atom, which suggests that the MoFe protein has two EPR active centers. Quantitative evaluation of M?ssbauer spectra shows that approximately 8 iron atoms give rise to one quadrupole doublet; at lower temperatures magnetic spectra, associated with the groud electronic doublet, are observed; at least two magnetically inequivalent sites can be distinguished. Taken together the data suggest that each EPR center contains 4 iron atoms. The EPR and M?ssbauer data can only be reconciled if these iron atoms reside in a spin-coupled (S = 3/2) cluster. Under nitrogen fixing conditions the magnetic M?ssbauer spectra disappeared concurrently with the EPR signal and quadrupole doublets are obserced at all temperatures. The data suggest that each EPR active center is reduced by one electron. The M?ssbauer investigation reveals three other spectral components characteristic of iron nuclei in an environment of integer or zero electronic spin, i.e. they reside in complexes which are "EPR-silent". One of the components (3-4 iron atoms) has M?ssbauer parameters characteristic of the high-spin ferrous iron as in reduced ruberdoxin. However, measurements in strong fields indicate a diamagnetic environment. Another component, representing 9-11 iron atoms, seems to be diamagnetic also. It is suggested that these atoms are incorporated in spin-coupled clusters.     

Magnetic interactions in milk xanthine oxidase

The relaxation behavior of the EPR signals of MoV, FAD semiquinone, and the reduced Fe/S I center was measured in the presence and absence of other paramagnetic centers in milk xanthine oxidase. Specific pairs of prosthetic groups were rendered paramagnetic by poising the native enzyme or its desulfo glycol inhibited derivative at appropriate potentials and pH values. Magnetic interactions were found between the following species: Mo--Fe/S I (100-fold increase in microwave power required to saturate the MoV EPR signal at 103 K when Fe/S I is reduced as opposed to oxidized), FAD--Fe/S I and FAD--Fe/S II (70-fold increase in power required to saturate the FADH.EPR signal at 173 K when either Fe/S center is reduced), and Fe/S I--Fe/S II (2.5-fold increase in power to saturate the reduced Fe/S I EPR signal at 20 K when Fe/S II is reduced). The Mo--Fe/S I interaction was also detected as a reduced Fe/S I induced splitting of the MoV EPR spectrum at 30 K. No splittings of the FADH. or Fe/S center spectra were detected. No magnetic interactions were found between FAD and Mo or between Mo and Fe/S II. These results, together with those of Coffman & Buettner [Coffman, R. E., & Buettner, G. R. (1979) J. Phys. Chem. 83, 2392-2400], were used to estimate the following approximate distances between the electron carrying prosthetic groups of milk xamthine oxidase: Mo--Fe/S I, 11 +/- 3 A; Fe/S I-Fe/S II, 15 +/- 4 A; FAD-Fe/S I, 16 +/- 4 A; FAD-Fe/S II, 16 +/- 4 A. A model for the arrangement of these groups within the xanthine oxidase molecule is suggested.     

A purple acid phosphatase from sweet potato contains an antiferromagnetically coupled binuclear Fe-Mn center

A purple acid phosphatase from sweet potato is the first reported example of a protein containing an enzymatically active binuclear Fe-Mn center. Multifield saturation magnetization data over a temperature range of 2 to 200 K indicates that this center is strongly antiferromagnetically coupled. Metal ion analysis shows an excess of iron over manganese. Low temperature EPR spectra reveal only resonances characteristic of high spin Fe(III) centers (Fe(III)-apo and Fe(III)-Zn(II)) and adventitious Cu(II) centers. There were no resonances from either Mn(II) or binuclear Fe-Mn centers. Together with a comparison of spectral properties and sequence homologies between known purple acid phosphatases, the enzymatic and spectroscopic data strongly indicate the presence of catalytic Fe(III)-Mn(II) centers in the active site of the sweet potato enzyme. Because of the strong antiferromagnetism it is likely that the metal ions in the sweet potato enzyme are linked via a mu-oxo bridge, in contrast to other known purple acid phosphatases in which a mu-hydroxo bridge is present. Differences in metal ion composition and bridging may affect substrate specificities leading to the biological function of different purple acid phosphatases.     

EPR and M?ssbauer studies of protocatechuate 4,5-dioxygenase. Characterization of a new Fe2+ environment

Protocatechuate 4,5-dioxygenase from Pseudomonas testosteroni has been purified to homogeneity and crystallized. The iron containing, extradiol dioxygenase is shown to be composed of two subunit types (alpha, Mr = 17,700 and beta, Mr = 33,800) in a 1:1 ratio; such a composition has not been observed for other extradiol dioxygenases. The 4.2 K M?ssbauer spectrum of native protocatechuate 4,5-dioxygenase prepared from cells grown in 57Fe-enriched media consists of a doublet with quadrupole splitting, delta EQ = 2.22 mm/s, and isomer shift delta Fe = 1.28 mm/s, demonstrating a high spin Fe2+ site. These parameters, and the temperature dependence of delta EQ, are unique among enzymes but are strikingly similar to those reported for the reaction center of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26, suggesting very similar ligand environments. The Fe2+ of protocatechuate 4,5-dioxygenase can be oxidized, for instance by H2O2, to yield high spin Fe3+ with EPR g values around g = 6 (and g = 4.3). In the oxidized state, protocatechuate 4,5-dioxygenase is inactive; the iron, however, can be rereduced by ascorbate to yield active enzyme. Our data suggest that protocatechuate binds to Fe2+; the spectra indicate that the ligand binding is heterogenous. The M?ssbauer spectra observed here are fundamentally different from those reported earlier (Zabinski, R., Münck, E., Champion, P., and Wood, J. M. (1972) Biochemistry 11, 3212-3219). The spectra of the earlier (reconstituted) preparations, which had substantially lower specific activities, probably reflect adventitiously bound Fe3+. We discuss here how adventitiously bound iron can be identified and removed. The Fe2+ which is present in native protocatechuate 4,5-dioxygenase and its complexes with substrates and inhibitors reacts quantitatively with nitric oxide to produce a species with electronic spin S = 3/2. The EPR and M?ssbauer spectra of these complexes compare favorably with EDTA . Fe(II) . NO. We have studied the latter complex extensively and have analyzed the M?ssbauer spectra with an S = 3/2 spin Hamiltonian. EPR spectra show that protocatechuate 4,5-dioxygenase-NO complexes with substrates or inhibitors are heterogeneous and consist of several well defined subspecies. The data show that NO, and presumably also O2, has access to the active site Fe2+ in the enzyme-substrate complex. The use of EPR-detectable NO complexes as a rapid and sensitive tool for the study of the EPR silent active site iron of extradiol dioxygenases is discussed.     

Binding of 17O-labeled substrate and inhibitors to protocatechuate 4,5-dioxygenase-nitrosyl complex. Evidence for direct substrate binding to the active site Fe2+ of extradiol dioxygenases

Pseudomonas testosteroni protocatechuate 4,5-dioxygenase catalyzes extradiol-type oxygenolytic cleavage of the aromatic ring of its substrate. The essential active site Fe2+ binds nitric oxide (NO) to produce an EPR active complex with an electronic spin of S = 3/2. Hyperfine broadening of the EPR resonances of the nitrosyl complex of the enzyme by protocatechuate (3,4-(OH)2-benzoate, PCA) enriched specifically with 17O (I = 5/2) in either the 3 or the 4 hydroxyl group shows that both groups can bind directly to the Fe2+ in the ternary complex. Analogous results are obtained for PCA binding to catechol 2,3-dioxygenase-NO complex suggesting that substrate binding by the Fe2+ may be a general property of extradiol dioxygenases. The protocatechuate 4,5-dioxygenase inhibitor, 4-17OH-benzoate binds directly to the Fe of the nitrosyl adduct of the enzyme through the OH group. Since previous studies have shown that water also is bound to the Fe in this ternary complex, but not in the ternary complex with PCA, the data strongly imply that there are 3 sites in the Fe coordination which can be occupied by exogenous ligands. 3-17OH-benzoate is an inhibitor of the enzyme but does not elicit detectable hyperfine broadening in the EPR spectrum of the nitrosyl adduct suggesting that it binds to the enzyme, but not to the Fe. The EPR spectra of ternary enzyme-NO complexes with PCA or 4-OH-benzoate labeled with 17O exclusively in the carboxylate substituent are not broadened, suggesting that this moiety does not bind to the Fe.     

Use of spin-labelled substrate analogs for a comparative study of microsomal cytochrome P-450 and P-448

The previously described, iodine-labeled alkylating stable nitroxyl radicals located at different distances between the N-O. group and the iodine atom were used for a comparative study of the structure of microsomal cytochromes P-450 and P-448 active centers. The radicals were shown to change the optical spectra of Fe3+ located in the active site of the enzyme that are similar to those induced by cytochrome P-450 substrates. Some differences in the type of the radicals binding to control, phenobarbital- and 3-methylcholanthrene-induced microsomes were revealed. The alkylating radical substrate analogs covalently bound to microsomal cytochrome P-450 in the vicinity of the active center, resulting in the inhibition of oxidation of type I and II substrates (e. g., aniline and naphthalene). The value of the spectral binding constant (Ks) for naphthalene in the presence of the radical covalently bound to the cytochrome P-450 active center showed a tendency to increase. Using the ESR technique, the interaction between Fe3+ and the radical localized in the active site of cytochrome P-450 was demonstrated. The contribution of Fe3+ to the relaxation of the radicals covalently bound to cytochrome P-450 was evaluated from the values of the spin label ESR spectra saturation curves at 77K. The distances between the N-O. group of these radicals and Fe3+ in the enzyme active center for the three types of microsomes were determined. The data obtained point to structural peculiarities of the active center of cytochrome P-450, depending on the microsomal type.     

Purification and characterization of adrenal cortex mitochondrial cytochrome P-450 specific for cholesterol side chain cleavage activity.

Cytochrome P-450 was purified from bovine adrenal cortex mitochondria by affinity chromatography using an octylamine-substituted Sepharose column. The resulting optically clear preparation was stable at -20 degrees for months. The specific concentration of cytochrome P-450 in the preparation was about 5 nmol of heme per mg of protein. The preparations were free of adrenodoxin, adrenodoxin reductase, phospholipids, and other heme contaminations. Polyacrylamide gel electrophoresis of the purified cytochrome P-450 preparation treated with sodium dodecyl sulfate and mercaptoethanol showed a single major band with a molecular weight of about 60,000. The optical absorption spectra of the preparation exhibited Soret maxima at 416, 416, and 448 nm for the Fe3+, Fe2+ and the C.Fe2+ complex, respectively. The EPR spectrum showed the characteristic features of the low spin form of ferric cytochrome P-450 with principal components 1.914, 2.241, and 2.415 of the g-tensor. The circular dichroism spectrum revealed two large negative ellipticities at 412 and 350 nm. Fluorescence spectra showed an excitation maximum at 285 nm and an emission maximum at 305 nm with a shoulder at 330 nm as the cytochrome P-450 molecule is excited at 285 nm, or an emission maximum at 335 nm when the cytochrome molecule is excited at 305 nm. After reconstitution with adrenodoxin and its reductase, this cytochrome P-450 was highly active for cholesterol desmolase with an NADPH-generating system as electron donor but was not active for steroid 11beta-hydroxylase.     

17O]Water and nitric oxide binding by protocatechuate 4,5-dioxygenase and catechol 2,3-dioxygenase. Evidence for binding of exogenous ligands to the active site Fe2+ of extradiol dioxygenases

Pseudomonas testosteroni protocatechuate 4,5-dioxygenase and Pseudomonas putida catechol 2,3-dioxygenase (metapyrocatechase) catalyze extradiol-type oxygenolytic cleavage of the aromatic ring of their substrates. The essential active site Fe2+ of each enzyme binds nitric oxide (NO) to produce an EPR active complex with an electronic spin of S = 3/2. Hyperfine broadening of the EPR resonances of the nitrosyl complexes by 17O-enriched H2O shows that water is bound directly to the Fe2+ in the native enzymes, but is apparently displaced in substrate complexes. NO is not displaced by either substrates or inhibitors. The EPR spectra of several enzyme-inhibitor-NO complexes are different from those of enzyme-NO or enzyme-substrate-NO complexes and are found to be broadened by 17O-enriched water. The data show that at least 2 and perhaps 3 sites in the Fe ligation can be occupied by exogenous ligands. Furthermore, it is likely that substrates and inhibitors displace water by binding either at or near to the Fe in the nitrosyl complex. Nitric oxide binding is found to be substrate-dependent for each enzyme. Native catechol 2,3-dioxygenase exhibits KD values of 190 microM and 2.0 mM for NO binding in two types of independent sites. Only one type of site is observed in the catechol complex which exhibits a KD for NO of 3.4 microM. One type of NO binding site is observed for both the native and substrate complexed protocatechuate 4,5-dioxygenase with KD values of 360 and 3 microM, respectively. The presence of a specific site in the Fe coordination for NO which is modified in the substrate complex, suggests that O2 binding by the extradiol dioxygenases may also occur at the Fe.     

Spectroscopic characterization of polyethyleneglycol modified superoxide dismutase: 1H NMR studies on its Cu2Co2 derivative

Spectroscopic methods have been employed in order to understand the molecular basis of the decrease in enzymatic activity of the antiinflammatory enzyme copper-zinc superoxide dismutase (SOD) following the covalent binding of polyethyleneglycol (PEG) chains to the protein amino-groups. The PEG modification is a general method recently proposed to improve the therapeutic index of enzymes. 1H NMR spectra on the cobalt substituted PEG-modified SOD, Cu2Co2-PEG-SOD, have been recorded. The signals are quite broad with respect to the unmodified enzyme. This has been interpreted on the basis of the effect of molecular weight on the linewidth. The analysis has shown that the histidine hydrogens involved in metal binding at the enzyme active site are the same in both native and PEG-modified SOD. Similarly, circular dichroism and absorption spectra indicate that the overall conformation of the metal clusters is not perturbed upon modification. On the other hand, azide titration shows that the affinity constant of N-3 for SOD is largely reduced upon PEG modification (K = 154 M-1 and 75 M-1 for the native and modified SOD, respectively). These results indicate that the decrease in enzymatic activity upon surface modification with PEG is not caused by a perturbation of the active site geometry, but to a decrease in the channeling of the O2- ion towards the enzyme active site.     

The red light of the helium-neon laser reactivates superoxide dismutase

The effect of low-energy helium-neon laser (HNL) on enzymatic activity, absorbtion spectra and electron paramagnetic resonance (EPR) signals of superoxide dismutase (SOD) from bovine erythrocytes in acid medium were investigated. It was found that incubation during 2 hours at pH 5.9 led to eventually complete inactivation of the enzyme. The subsequent illumination of inactivated SOD by HNL brought about the enzyme reactivation. Both absorption and EPR-spectra were changed after incubation at pH 5.9. These changes may be attributed to protonation of histidine residue in the enzyme active site. After laser irradiation both absorption and EPR spectra were restored to those typical of native enzyme at pH 8.2. In a model system, copper-histidine complex, absorption maximum was shifted from 632-633 nm at pH 5.8 to 639-640 nm at pH 8.5-9.0. The similar long-wave length shift of the maximum was observed after illumination by HNL at pH 5.8. It may be postulated that the photoreactivation of SOD consists essentially in deprotonation of His-61 residue in the enzyme active site and subsequent recovery of imidazole bridge between copper and zinc which had been destroyed at low pH. Since many other enzymes possess similar copper-histidine structures in their active sites, one may expect diverse effects of red (laser) light on the enzyme activity.