ESR signals of photosystem II after complete removal of manganese from pea subchloroplast particles

The effect of reversible extraction of Mn on ESR signal II arising from the oxidized secondary electron donor (Z+) and the ESR doublet signal related to reduced spin-coupled pheophytin (pheo -) and "primary" electron acceptor (PA- -Fe (2+)) has been studied in oxygen evolving preparations of the photosystem 2. It is demonstrated that Mn extraction does not affect both dark and photoinduced ESR signal II and ESR doublet. A conclusion is made that Mn is not a component of the secondary electron donor Z of the Photosystem 2 and its complete removal has no effect on the exchange interaction of Pheo(-) and the PQ(-) -Fe(2+) complex.     

Functions of pheophytin, plastoquinone, iron and carotenoids in plant photosystem 2 reaction centers

Photoreduction of the intermediary electron acceptor, pheophytin (Ph), in photosystem-2 (PS-2) reaction centers of spinach chloroplasts or subchloroplast particles (TSF-II and TSF-IIa) at 220 K and Eh approximately -450 mV produces a narrow ESR signal of Ph. (g = 2.0033; delta H approximately 13 G) and a "doublet" centered at g = 2.00 with a splitting of 52 G at 7 K. The doublet (but not the narrow signal) is eliminated after extraction of lyophylized TSF-II with hexane, containing 0.1-0.2% methanol, or after extraction of Fe with LiClO4 and o-phenantroline, and the signal is restored by reconstitution with plastoquinone-A (PQ) or Fe++, respectively. The Fe removal results also in the development of a photoinduced ESR signal of PQ. (g approximately 2.0044; delta H approximately 9.2 G). The conclusion is made that the primary electron acceptor, Q, is in fact a complex PQ-Fe++ and that the exchange interaction of Ph. with PQ. -Fe++ is responsible for the ESR doublet. Photoreduction of Ph in TSF-IIa is accompanied by the 3-fold decrease in the formation of carotenoid triplet state (measured by the characteristic flash-induced absorbance changes) which is suggested to be a result of charge recombination in the pair [P680+ .PH.].     

Identification of A Free Radical and Oxygen Dependence of Ribonucleotide Reductase in Yeast

Ribonucleotide reductase is a key enzyme for DNA biosynthesis. The enzymes isolated from animal and plant cells possess a stable tyrosyl free radical which is essential for catalysis. Fungal ribonucleotide reductases are little known; the partially characterized enzyme from yeast cells proved exceptionally shortlived, and a free radical could not as yet be demonstrated. We here show that a doublet ESR signal centered at g = 2.0046 can be measured below 60°K in rapidly purified protein samples which is very similar to the ESR spectra of the tyrosine radicals present in other eukaryotic ribonucleotide reductases in structure, microwave saturation, and quenching by hydroxyurea. Because generation of these radicals requires oxygen, anaerobic yeast cultures were also studied. No change in ribonucleotide reductase was observed at 50ppm residual oxygen in the gas phase, but cell proliferation ceased entirely under complete anaerobiosis.     

Identification of A Free Radical and Oxygen Dependence of Ribonucleotide Reductase in Yeast

Ribonucleotide reductase is a key enzyme for DNA biosynthesis. The enzymes isolated from animal and plant cells possess a stable tyrosyl free radical which is essential for catalysis. Fungal ribonucleotide reductases are little known; the partially characterized enzyme from yeast cells proved exceptionally shortlived, and a free radical could not as yet be demonstrated. We here show that a doublet ESR signal centered at g = 2.0046 can be measured below 60°K in rapidly purified protein samples which is very similar to the ESR spectra of the tyrosine radicals present in other eukaryotic ribonucleotide reductases in structure, microwave saturation, and quenching by hydroxyurea. Because generation of these radicals requires oxygen, anaerobic yeast cultures were also studied. No change in ribonucleotide reductase was observed at 50ppm residual oxygen in the gas phase, but cell proliferation ceased entirely under complete anaerobiosis.     

Nonlineal relationship between g=2 doublet and multiline signals in Ca(2+)-depleted Photosystem II

Illuminating of the Ca(2+)-depleted PS II in the S(2) state for a short period induced the doublet signal at g=2 with concomitant diminution of the multiline signal, both in the presence and absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). In the absence of DCMU, the doublet signal decayed (t(1/2) approximately 7 min) during subsequent dark incubation at 273 K and the multiline signal was regenerated to the original amplitude with the same kinetics of the doublet decay. In the presence of DCMU, the doublet signal decayed much faster (t(1/2) approximately 1 min) by charge recombination with Q(A)(-), while the time course of the multiline recovery was inherently identical with that observed in the absence of DCMU. A simple theoretical consideration indicates the direct conversion from the doublet-signal state to the multiline state with no intermediate state between them. Lengthy dark storage at 77 K led to disappearance of the DCMU-affected doublet signal and a Fe(2+)/Q(A)(-) electron spin resonance (ESR) signal, but no recovery of the multiline signal. Notably, the multiline signal was restored by subsequent dark incubation at 273 K. The charge recombination between Q(A)(-) and the doublet signal species led to a thermoluminescence band at 7 degrees C in a medium at pH 5.5. The peak position shifted to 17 degrees C at pH 7.0, presumably due to a pH-dependent change in the redox property of a donor-side radical species responsible for the doublet signal. Based on these results, redox events in the Ca(2+)-depleted PS II are discussed in contradistinction with the normal processes in oxygen-evolving PS II.     

Purification and properties of methanol dehydrogenase from Hyphomicrobium x.

(1) A method for the isolation of methanol dehydrogenase (alcohol:(acceptor) oxidoreductase, EC 1.1.99.8) from Hyphomicrobium X is decribed. The purified enzyme was resolved by polyacrylamide gel electrophoresis into one main and two minor active bands. Iron and manganese were the only detected metals in the enzyme preparation. (2) The substrate, methanol, was oxidized to formic acid by a stoichiometric amount of artificial electron acceptor. During the reaction, no free formaldehyde could be detected. Other primary alcohols were oxidized to the corresponding aldehydes were a poor substrate or no substrate at all. (3) Some new and efficient one-electron acceptors were found. With these electron acceptors, the enzyme had a high pH optimum and ammonia was still required in the assay system. (4) ESR spectroscopy showed the presence of an enzyme-bound organic free radical. With X-band ESR the signal had a peak-to-peak linewidth of about 0.7 mT. The signal was further resolved by Q-band ESR and the values gparallel = 2.0024 and gperpendicular = 2.0056 were derived. (5) Under denaturing conditions the ESR signal and enzymatic activity disappeared at the same time as fluorescence appeared. Enzymatic activity is not restored when extracted cofactor and apoenzyme are brought together under normal conditions. Some properties of the unusual prosthetic group are presented in a preliminary form.     

Higher oxidation states of prostaglandin H synthase. EPR study of a transient tyrosyl radical in the enzyme during the peroxidase reaction

Purified prostaglandin H synthase (EC 1.14.99.1), reconstituted with hemin, was reacted with substrates of the cyclooxygenase and peroxidase reaction. The resulting EPR spectra were measured below 90 K. Arachidonic acid, added under anaerobic conditions, did not change the EPR spectrum of the native enzyme due to high-spin ferric heme. Arachidonic acid with O2, as well as prostaglandin G2 or H2O2, decreased the spectrum of the native enzyme and concomitantly a doublet signal at g = 2.005 was formed with maximal intensity of 0.35 spins/enzyme and a half-life of less than 20 s at -12 degrees C. From the conditions for the formation and the effect of inhibitors, this doublet signal was assigned to an enzyme intermediate of the peroxidase reaction, namely a higher oxidation state. The doublet signal with characteristic hyperfine structure was nearly identical to the signal of the tyrosyl radical in ribonucleotide reductase (EC 1.17.4.1). Hence the signal of prostaglandin H synthase was assigned to a tyrosyl radical. Electronic spectra as well as decreased power saturation of the tyrosyl radical signal indicated heme in its ferryl state which coupled to the tyrosyl radical weakly. [FeIVO(protoporphyrin IX)]...Tyr+. was suggested as the structure of this two-electron oxidized state of the enzyme. A hypothetical role for the tyrosyl radical could be the abstraction of a hydrogen at C-13 of arachidonic acid which is assumed to be the initial step of the cyclooxygenase reaction.     

Room temperature electron spin resonance of superoxide dismutase-loaded liposomes and erythrocytes. A direct approach to the interaction of O2- with cells

Human erythrocytes were enriched with bovine superoxide dismutase by fusion with liposomes containing the entrapped enzyme. Liquid solution ESR of intact cells at room temperature was used to measure directly the increase in the superoxide dismutase content. From the spectral characteristics (g-value and hyperfine splitting tensor), the structural integrity of the Cu site of the enzyme was found to be unaffected by the liposome preparation procedure or the incubation with cells. Changes in the ESR signal size were used to test directly the interaction of superoxide with the enzyme entrapped in liposomes or delivered to erythrocytes. It was found that the liposome-entrapped enzyme does not react with externally generated O2-, but once delivered to red blood cells this reaction can take place. This is the first demonstration of O2- -scavenging activity by superoxide dismutase delivered into an intact cell structure and is therefore to be considered as strong evidence for activity of this enzyme under in vivo conditions.     

Site-directed mutagenesis of a possible type 1 copper ligand of bilirubin oxidase; a Met467Gln mutant shows stellacyanin-like properties

In our previous paper, we reported a mutant of recombinant Myrothecium verrucaria bilirubin oxidase, in which the Met467 residue was replaced by Gly [Shimizu, A. et al. (1999) Biochemistry 38, 3034-3042]. This mutant displayed a remarkable reduction in enzymatic activity and an evident decrease in the intensity of the absorption band around 600 nm (type 1 charge transfer transition). In this study, we report the preparation of three Met467 mutants (Met467Gln, Met467His, and Met467Arg) and characterize their enzymatic activities, midpoint potentials, and absorption and ESR spectra. Met467His and Met467Arg show no enzymatic activity and a great reduction in the intensity of the absorption band around 600 nm. Furthermore, their ESR spectra show no type 1 copper signal, but only a type 2 copper signal; however, oxidation by ferricyanide caused the type 1 copper signal to appear. On the other hand, Met467Gln as expressed shows both type 1 and type 2 copper signals in its ESR spectrum, the type 1 copper atom parameters being very different from usual blue copper proteins but very similar to those of stellacyanin. The enzymatic activity of the Met467Gln mutant for bilirubin is quite low (0.3%), but the activity for potassium ferrocyanide is similar (130%) to that of the wild type enzyme. These results indicate that Met467 is important for characterizing the features of the type 1 copper of bilirubin oxidase.     

Metallobleomycins: electron spin resonance study on nitrosyl complex of iron(II)-Bleomycin

The ESR spectrum of the bleomycin-Fe(II)NO complex shows rhombic symmetry with a triplet hyperfine interaction in the g_z signal, and its ESR parameters have been compared with those of the ferrous-NO complexes of hemoproteins. The substitution of ₁₄NO by ₁₅NO gives the transition from a triplet to a doublet in the g_z absorption with a concomitant change in the nitrogen hyperfine constant. The addition of DNA to the ferrous-NO complex of bleomycin induces the greater separation of the g_x and g_y absorptions in comparison with the original ESR spectrum. The present three-line g_z signal for the bleomycin-Fe(II)-NO complex is indicative of weakened fifth axial nitrogen ligand-to-iron bonding with concomitantly stronger NO-to-iron bonding. On the other hand, the ESR feature of the bleomycin-Fe(III) complex is typical of the rhombic low-spin type, and no stable ferric-NO complex of bleomycin is formed.     

ESR-detectable nickel and iron-sulphur centres in relation to the reversible activation of Desulfovibrio gigas hydrogenase

The electron spin resonance (ESR) spectra of hydrogenase from Desulfovibrio gigas were observed during the activation of the enzyme in the oxidized, ‘unready’, state by hydrogen. Signals from nickel(III) (Ni-A), and the [3Fe-xS] cluster were reduced within less than 5 min, and a broad ESR signal appeared at the same time. On the basis of simultaneous changes in optical absorption spectrum, it is proposed that the broad ESR signal represents one or possibly both [4Fe-4S] clusters in the reduced state. The increase of enzyme activity was much slower (at 20°C), and was accompanied by the appearance of another type of nickel signal (Ni-C), and a further small decrease and the Ni-C signal became more intense. On further reoxidation by the dye dichlorophenolindophenol at pH above 7.0 the enzyme was converted to the ‘ready’ state, which could now be reactivated much more rapidly by strong reductants. The proportion of the ready state correlated with a third type of nickel signal, Ni-B. The unready enzyme could also be slowly activated by milder reducing conditions which reduced Ni-A and the [3Fe-xS] cluster but did not induce significant amounts of the Ni-C and [4Fe-4S]¹⁺ signals. The optical absorption changes indicate that the Ni-A is not coupled to an iron-sulphur cluster. It is proposed that the activation of the enzyme involves reduction of the nickel and possibly iron-sulphur centres, followed by a conformational change which alters the coordination state of nickel, and that the unready state contains Ni(III) in the inactive conformation, the ready state Ni(III) in the active conformation, and the active state Ni(I).     

Leukemia and the reducing property of viruses

Summary Investigations of native blood of healthy people and of patients with acute leukemia have shown that the disease might be caused by a strongly reducing substance, which is presumably a virus. Evidence for this conclusion was obtained mainly by electron spin resonance (ESR) studies and by the determination of the catalase activity. ESR spectra of leukemic blood revealed an additional signal not present in spectra of healthy blood. Investigation of different blood fractions has shown that this signal is caused by a species present in the leukocytes only. Addition of reduced glutathione to healthy blood immediately after blood drawing resulted in the same signal. On the contrary, oxidizing substances, such as oxidized glutathione and KMnO₄, added to the blood of leukemic patients immediately after its drawing, caused a disappearance of this signal depending on the concentration.Since the strongly reducing substance causes a reduction in the concentration of the oxidizing substances in biological systems, the H₂O₂ level should be also affected and, thus, the catalase activity, too. As expected, the activity of this enzyme in the leukemic blood seems to be considerably lower than in healthy blood.     

Free radical involvement in long wavelength UV light activation of nitrosamines to mutagens

Nitrosamines are carcinogenic and mutagenic only after metabolic activation via endoplasmic reticulum bound mixed function oxidase enzyme systems. Rencently a new photochemical process has been discovered by which nitrosamines are converted into unknown mutagenic compounds by irradiation with long wavelength UV light (> 335 nm) in the presence of phosphate ion at neutral pH. The mutagenic activity is detected by Ames Salmonella Typhimurium strain TA100 in the absence of rat liver microsomes. We have shown that mutagen production with nitrosomorpholine is inhibited in the presence of light by various spin trapping agents (N-t-butyl-phenylnitrone, etc.). Concurrent with this inhibition a stable free radical signal has been detected whose kinetics of formation is similar to the time course of mutagen formation during irradiation in the absence of spin trap. The free radical signal is formed only when phosphate or similar ions are present in the reaction mixture. Monomethylphosphate and dimethylphosphate can substitute for phosphate ion but with small ESR signals and mutagen formation. Trimethylphosphate gives a weak, time independent ESR signal and does not cause mutagen formation. The ESR splitting constants (a^N and a^H) for signals generated with each of the different phosphate species show differences which suggest that these ions may be components of some intermediate free radical species that is involved in stable mutagen formation. Arsenate ion inhibits mutagen formation in the presence of phosphate but is able in the absence of phosphate to form a ESR signal similar to that observed with phosphate ion.     

Electron spin resonance investigation of tyrosyl radicals of prostaglandin H synthase. Relation to enzyme catalysis.

We have examined, by low temperature ESR, the protein-derived radicals formed by reaction of purified ram seminal vesicle prostaglandin H synthase (PHS). Upon addition of arachidonic acid or 5-phenyl-4-pentenyl-1-hydroperoxide (PPHP) to PHS reconstituted with Fe(III)-protoporphyrin IX (Fe-PHS) at -12 degrees C, an ESR spectrum was observed at -196 degrees C containing a doublet that rapidly converted into a singlet. These protein-derived radicals were identified as tyrosyl radicals. The addition of a peroxidase substrate, phenol, completely abolished the appearance of the doublet and suppressed the formation of the singlet but did not inhibit eicosanoid formation. Incubation of arachidonic acid with PHS reconstituted with Mn(III)-protoporphyrin IX (Mn-PHS) produced only a broad singlet that exhibited different power saturation behavior than the tyrosyl radicals and decayed more rapidly. This broad singlet does not appear to be a tyrosyl radical. No ESR signals were observed on incubation of PPHP with Mn-PHS, which has cyclooxygenase but not peroxidase activity. Eicosanoid synthesis occurred very rapidly after addition of arachidonic acid and was complete within 1 min. In contrast, the protein-derived radicals appeared at a slower rate and after the addition of the substrate reached maximal levels between 1 and 2 min for Fe-PHS and 4-6 min for Mn-PHS. These results suggest that the observed protein-derived radicals are not catalytically competent intermediates in cyclooxygenase catalysis by either Fe-PHS or Mn-PHS. The peroxidase activity appears to play a major role in the formation of the tyrosyl radicals with Fe-PHS.     

NAD-dependent hydrogenase from Alcaligenes eutrophus Z1: Does it have a regulatory centre?

Evidence is presented for the existence of a relatively high-potential regulatory centre in the NAD-dependent hydrogenase from the hydrogen oxidizing bacterium Alcaligenes eutrophus Z1. Reduction of the hydrogenase to the redox potentials lower than -100 mV converts the enzyme into a catalytically active state that is remarkably stable to oxidants. Once activated, the enzyme does not loose its activity on intensive oxygenation for at least 3 hours. A novel hydrogenase ESR signal with a wide temperature optimum and a approximately -100 mV midpoint redox potential was detected. We suggest that the reduction of this redox centre trigger conformational changes in the inactive oxidized enzyme molecule, thus reorganizing the latter into the active one.     

Study of the binding of substrates and paramagnetic Mn2+ ions with phosphoglycerate kinase by saturating ESR spectra of a spin-labelled protein]

A single SH-group of phosphoglycerate kinase from yeast was modified by mercury-containing spin label. The saturation curves of ESR spectra of the spin-labeled enzyme were studied. The paramagnetic ions of Mn2+ bound to the centre of ion nonspecific binding or active centre in the complex with ATP can influence the saturation of the spin-labeled enzyme. The saturation curves of the ESR signal of the spin-labeled enzyme in the presence of paramagnetic complex of CrATP were studied. It has been demonstrated that the second nonspecific centre of ATP binding is located at the active site of the enzyme (3-phosphoglycerate binding centre).     

Content and localization of FMN, Fe-S clusters and nickel in the NAD-linked hydrogenase of Nocardia opaca 1b

By preparative polyacrylamide gel electrophoresis at pH 8.5, and in the absence of nickel ions, two types of subunit dimers of the NAD-linked hydrogenase from Nocardia opaca 1b were separated and isolated, and their properties were compared with each other as well as with the properties of the native enzyme. The intact hydrogenase contained 14.3 +/- 0.4 labile sulphur, 13.6 +/- 1.1 iron and 3.8 +/- 0.1 nickel atoms and approximately 1 FMN molecule per enzyme molecule. The oxidized hydrogenase showed an absorption spectrum with maxima (shoulders) at 380 nm and 420 nm and an electron spin resonance (ESR) spectrum with a signal at g = 2.01. The midpoint redox potential of the Fe-S cluster giving rise to this signal was +25 mV. In the reduced state, hydrogenase gave characteristic low-temperature (10-20 K) and high-temperature (greater than 40 K) ESR spectra which were interpreted as due to [4Fe-4S] and [2Fe-2S] clusters, respectively. The midpoint redox potentials of these clusters were determined to be -420 mV and -285 mV, respectively. The large hydrogenase dimer, consisting of subunits with relative molecular masses Mr, of 64000 and 31000, contained 9.9 +/- 0.4 S2- and 9.3 +/- 0.5 iron atoms per protein molecule. This dimer contained the FMN molecule, but no nickel. The absorption and ESR spectra of the large dimer were qualitatively similar to the spectra of the whole enzyme. This dimer did not show any hydrogenase activity, but reduced several electron acceptors with NADH as electron donor (diaphorase activity). The small hydrogenase dimer, consisting of subunits with Mr of 56000 and 27000, was demonstrated to have substantially different properties. For iron and labile sulphur average values of 3.9 and 4.3 atoms/dimer molecule have been determined, respectively. The dimer contained, in addition, about 2 atoms of nickel and was free of flavins. In the oxidized state this dimer showed an absorption spectrum with a broad band in the 400-nm region and a characteristic ESR signal at g = 2.01. The reduced form of the dimer was ESR-silent. The small dimer alone was diaphorase-inactive and did not reduce NAD with H2, but it displayed high H2-uptake activities with viologen dyes, methylene blue and FMN, and H2-evolving activity with reduced methyl viologen. Hydrogen-dependent NAD reduction was fully restored by recombining both subunit dimers, although the reconstituted enzyme differed from the original in its activity towards artificial acceptors and the ESR spectrum in the oxidized state.     

Electron spin resonance spectra of free radicals formed in the reaction of metmyoglobins with ethylhydroperoxide

Free radicals of myoglobins were measured at room temperature with an ESR spectrometer equipped with a flow apparatus. When horse heart MetMb was mixed with an equimolar amount of ethyl hydroperoxide (EtOOH), a well resolved ESR spectrum with 6 lines and a shoulder was observed. It reached a maximum in a few seconds and decayed with a half-life of about 10 s when the final concentrations of MetMb and EtOOH were 200 microM. This decay rate was the same at a MetMb concentration of 50 microM. The maximum molar radical concentration amounted to about half of the total myoglobin. In the case of sperm whale myoglobin, a similar 6-line spectrum reached a maximum in 1 s and decayed with a half-life of a few seconds. In this case, however, a small and poorly resolved doublet spectrum remained, the half-life of which was about 8 min. An effect of O2 on the signal decay was evident for horse heart myoglobin, but not for sperm whale myoglobin.     

Pullulanase in mung bean cotyledons. Purification, some properties and developmental pattern during and following germination

Starch debranching enzyme was purified from mung bean ( Vigna radiata ) cotyledons to investigate its properties and developmental pattern during and following germination. A debranching enzyme was purified up to the step where only a doublet of polypeptides with molecular masses of 99 and 101 kDa, respectively, was detected by SDS-PAGE. The enzyme is thought to be a single chain monomer, as the molecular mass of the enzyme determined by gel filtration was 72 kDa. Monoclonal antibodies raised against the purified preparation recognized the doublet, indicating that the two polypeptides have immunological homology to each other. The enzyme preparation showed a high activity with pullulan as a substrate, low activity with soluble starch and amylopectin, and no activity with glycogen. These substrate specificities indicate that the debranching enzyme from mung bean cotyledons is of the pullulanase type. Immunoblotting profiles revealed that the enzyme is present in dry seeds and decreases gradually after imbibition, suggesting the possibility that the pullulanase plays a role in developing mung bean cotyledons.     

In utero and in vitro proteinase activity during the Mesocricetus auratus embryo zona escape time window

The goal of the present study was to investigate proteinase activity in uterine flushates collected during the zona loss time window (68-80 h post-egg activation) in both pregnant and pseudopregnant hamsters and in culture medium conditioned by hatching blastocysts. Several prominent enzyme activities appeared in all pregnant and pseudopregnant uterine flushates. However, only a 45, 43 x 10(-3) M:(r) doublet coincided with the zona loss time window; these bands were absent outside of this time window and were not found in conditioned medium. In medium conditioned by hatching blastocysts, enzyme activity was represented by a 70, 65 x 10(-3) M:(r) doublet identical to a doublet seen in all uterine flushates collected and in serum. There were 12 pregnant and 8 pseudopregnant uterine flushates that were capable of zona lytic activity in vitro (positive bioassays). Of these positive bioassays, five pregnant and four pseudopregnant uterine flushates exhibited the 45, 43 x 10(-3) M:(r) doublet (correlative positive bioassays). These data suggest that there is an important uterine contribution to blastocyst escape from the zona pellucida, consisting of proteinases secreted during a finite time window prior to blastocyst attachment that are different from the proteinases responsible for the zona lytic activity in vitro.