Electron paramagnetic resonance identification of a highly reactive thiol group in the proximity of the catalytic site of human placenta glutathione transferase.

The reaction of the glutathione transferase from human placenta with a maleimide spin label derivative has been followed by EPR. Incubation of the enzyme at pH 7.0 with 50-fold molar excess of the spin label reagent gives rise to an immobilized nitroxyl EPR spectrum indicative of two reacting thiol groups per dimer of enzyme as evaluated by double integration of the EPR spectrum; the activity is lost in parallel. The same type of spectrum can be obtained simply by adding 2 eq of the spin label reagent to the enzyme. The binding is completed after less than 1 min at pH 8.0; it requires 2 min at pH 7.0 and more than 10 min at pH 6.0. These data indicate that the maleimide derivative reacts, in each subunit, with a thiol group which plays a crucial role for the maintenance of the catalytic activity and is characterized by a low pK. Inactivation of the enzyme at pH 7.0 in the presence of 2 eq of spin label reagent per mol of enzyme requires 15 min, suggesting the occurrence of a structural rearrangement after the binding of the thiol blocking agent. The same binding in the presence of S-methylglutathione or protoporphyrin IX shows a decreased reaction rate with respect to the reaction in the absence of inhibitors, indicating that the thiols are in proximity of both the glutathione and the porphyrin binding sites. For this latter case, this is unambiguously demonstrated by the titration of spin-labeled enzyme with hemin, which produces a decrease of the EPR signal amplitude from which an interspin distance of about 10 A can be evaluated.     

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.     

Identification of the iron entry channels in apoferritin. Chemical modification and spectroscopic studies

The knowledge of the route through which iron can enter and leave the apoferritin shell is a prerequisite for the understanding of ferritin's function. The involvement of the hydrophilic 3-fold channels in the iron uptake process has been studied by taking advantage of the reactivity of specific residues that line such channels, i.e., glutamic acid-127 and aspartic acid-130, the major Cd(II) binding sites, and cysteine-126. 113Cd NMR experiments have provided direct evidence for the competition between Fe(II) and Cd(II) binding to major Cd(II) binding sites on the protein and or a higher affinity of Fe(II) for these sites, in line with the well-known inhibitory effect of Cd(II) on iron uptake. Further evidence for the use of the 3-fold channels in the iron entry process has been obtained by means of chemical modification of Cys-126 with different mercurials. In particular, the introduction of the additional carboxylate carried by p-(chloromercuri)benzoate near Asp-127 and Glu-130 increases the initial rate of iron uptake and affects the coordination geometry of the metal in the Fe(III)-apoferritin complex as indicated by optical absorption and EPR data. The assignment of these effects to the carboxylate moiety of p-(chloromercuri)benzoate is brought out by the observation that the introduction in the 3-fold channel of the benzene ring only by means of phenylmercuric acetate has no effect on the initial iron uptake kinetics and on the spectroscopic properties of the Fe(III)-apoferritin complex.     

Photoreduction of copper chromophores in blue oxidases.

The low temperature (77 K) irradiation of oxidized ceruloplasmin and Rhus vernicifera laccase at the 330 nm absorption which arises from type 3 copper leads to the reduction of type 1 copper as demonstrated by bleaching of the 610 nm chromophore and the decrease of the EPR signal associated with this species. Type 2 copper remains unaffected. Concomitant with the type 1 copper reduction, a new EPR signal which is possibly that of a biradical appears. Upon thawing, type 1 copper is reversibly oxidized and the radical signal disappears. Irradiation of oxidized protein at the absorption band of type 1 copper produces no spectral change. An EPR study at room temperature confirms the wave-length specificity and reversibility of the photoreduction of type 1 copper and radical formation. Radical appearance and disappearance at room temperature are extremely slow (tau1/2 approximately 30 min). Optical studies at room temperature show that upon anaerobic irradiation of laccase in the 330 nm absorption band, both type 3 and type 1 chromophores are slowly reduced. Upon return to the dark and in the presence of O2, both type 3 and type 1 centers are reoxidized. Oxidizing equivalents either from O2 or K3Fe(CN)6 are required for the reoxidation reaction. These studies demonstrate that there is a direct energy transfer between type 3 and type 1 copper sites in blue copper oxidases.     

Cholesterol-induced alterations of the packing properties of gangliosides: an EPR study

The effect of cholesterol (Chol) on two kinds of glycolipid assemblies, one composed of monosialogangliosides (GM1a) and the other formed by a natural mixture of bovine brain gangliosides (TBG), has been analysed. The experimental approach involves spin label electron paramagnetic resonance (EPR) in aqueous lipid dispersions. The employment of a hydrosoluble spin label and a 'quencher' of the EPR signal that is not able to permeate lipid interfaces, allowed us to conclude that GM1a/Chol mixtures give rise to vesicles at Chol proportions for which TBG/Chol mixtures form micelles. The use of different liposoluble spin labels reveals that cholesterol produces a straightening of the hydrocarbon chains in both lipid systems. In GM1a/Chol mixtures, this feature is more pronounced and it is coupled with a decrease in polarity at the chain ends.     

Metal ion complexes of apoferritin. Evidence for initial binding in the hydrophilic channels

Metal binding to the iron storage protein apoferritin is the first step in the process by which iron accumulates within the protein shell. In the present study, the stoichiometry of metal binding to apoferritin in solution has been examined using the probe ions Mn(II), VO(IV), and Cd(II) in conjunction with EPR spectroscopic and cadmium ion selective electrode measurements. Binding studies were carried out with the individual ions, in competition with one another, and in competition with Fe(II), Fe(III), and Tb(III). All three probe ions show binding stoichiometries near 0.3 and 0.7 metal ion per subunit, close to the theoretically predicted values of 0.33 and 0.67 for the binding of one and two metal ions, respectively, per three subunits. These results in conjunction with other data are consistent with the binding of one, and possibly two, metal ions within each of the eight hydrophilic channels which are located on 3-fold axes leading to the interior of the protein. Pairs of cadmium binding sites have been located in these channels by x-ray crystallography (Rice, D. W., Ford, G. C., White, J. L., Smith, J. M. A., and Harrison, P. M. (1983) Adv. Inorg. Biochem. 5, 39-49). The possibility that some metal binding occurs elsewhere on the protein is not precluded by the present data, however. In competition experiments between various metal ions, approximately 0.3 metal ion per subunit is readily displaced implying common binding sites in the channels for all of them. The stoichiometry of Mn(II) displacement by Fe(II) is less clear. Oxidation of Fe(II) to Fe(III) by molecular oxygen in the presence of Mn(II) regenerates some Mn(II) binding on the protein, suggesting migration of iron(III) to other protein sites, or perhaps to core.     

Photoinhibition affects the non-heme iron center in photosystem II.

Effects on the PS II acceptor side caused by exposure to strong white light (180 W/m2) of PS II membrane fragments (spinach) at pH 6.5 and 0 degrees C were analyzed by measuring low temperature EPR signals and flash-induced transient changes of the fluorescence quantum yield. The following results were obtained: (a) the extent of the light induced g = 1.9 EPR signal as a measure of photochemical Fe2+QA- formation declines with progressing photoinhibition. The half-life of this effect is independent of the absence or presence of an exogenous electron acceptor during the photoinhibitory treatment; (b) in samples photoinhibited in the absence of an electron acceptor and subsequently incubated with K3[Fe(CN)6] in the dark, the extent of the g = 8 EPR signal (reflecting the oxidized Fe3+ form of the endogenous non-heme iron center) and of the flash-induced change of the fluorescence yield (as a measure of fast electron transfer from QA- to Fe3+ after the first flash; [see (1992) Photosynth. Res. 31, 113-126] exhibits the same dependence on photoinhibition time as the g = 1.9 EPR signal; (c) in samples photoinhibited in the presence of an exogenous electron acceptor, the signals reflecting Fe(3+)-formation and fast electron transfer from QA- to Fe3+ decline faster than the g = 1.9 EPR signal. These results provide for the first time direct evidence that the endogenous non-heme iron center located between QA and QB is susceptible to modifications by light stress. The implications of this finding will be discussed.     

Detection of a [3Fe-4S] cluster intermediate of cytosolic aconitase in yeast expressing iron regulatory protein 1. Insights into the mechanism of Fe-S cluster cycling.

Interconversion of iron regulatory protein 1 (IRP1) with cytosolic aconitase (c-aconitase) occurs via assembly/disassembly of a [4Fe-4S] cluster. Recent evidence implicates oxidants in cluster disassembly. We investigated H(2)O(2)-initiated Fe-S cluster disassembly in c-aconitase expressed in Saccharomyces cerevisiae. A signal for [3Fe-4S] c-aconitase was detected by whole-cell EPR of aerobically grown, aco1 yeast expressing wild-type IRP1 or a S138A-IRP1 mutant (IRP1(S138A)), providing the first direct evidence of a 3Fe intermediate in vivo. Exposure of yeast to H(2)O(2) increased this 3Fe c-aconitase signal up to 5-fold, coincident with inhibition of c-aconitase activity. Untreated yeast expressing IRP1(S138D) or IRP1(S138E), which mimic phosphorylated IRP1, failed to give a 3Fe signal. H(2)O(2) produced a weak 3Fe signal in yeast expressing IRP1(S138D). Yeast expressing IRP1(S138D) or IRP1(S138E) were the most sensitive to inhibition of aconitase-dependent growth by H(2)O(2) and were more responsive to changes in media iron status. Ferricyanide oxidation of anaerobically reconstituted c-aconitase yielded a strong 3Fe EPR signal with wild-type and S138A c-aconitases. Only a weak 3Fe signal was obtained with S138D c-aconitase, and no signal was obtained with S138E c-aconitase. This, paired with loss of c-aconitase activity, strongly argues that the Fe-S clusters of these phosphomimetic c-aconitase mutants undergo more complete disassembly upon oxidation. Our results demonstrate that 3Fe c-aconitase is an intermediate in H(2)O(2)-initiated Fe-S cluster disassembly in vivo and suggest that cluster assembly/disassembly in IRP1 is a dynamic process in aerobically growing yeast. Further, our results support the view that phosphorylation of IRP1 can modulate its response to iron through effects on Fe-S cluster stability and turnover.     

Transition metal ion complexes with associates of porphyrin bound to a hydrophobic-hydrophilic copolymer.

The interaction of dimeric forms of meso-tetraphenylporphine with Mn2+ as well as the interaction of associated forms of meso-tetra(p-aminophenyl)porphine bound to a hydrophobic-hydrophilic copolymer with Mn2+ and Fe3+ were studied by absorption, luminescence, Raman, and EPR spectroscopies. Both dimeric and associated forms of these porphyrins produced Mn2+ complexes. Manganese ions in these complexes undergo clusterization, which is accompanied by transformation of the six-line EPR signal of Mn2+ into a broad single-line signal. The EPR signal of Mn2+ in these clusters is characterized by a g-factor value typical of a free electron with half-width DeltaHpp = 50 mT. The interaction of the two-component complex with Fe3+ produces a donor-acceptor complex. The electronic spectrum of the donor-acceptor complex contains a broad band with a maximum at 760 nm. The molar extinction coefficient of the complex at 760 nm is 9.1.104 M-1.cm-1, and the rate constant for its formation is Kdac = (3.9 +/- 0.6).106 M-1. The constants for Mn2+ binding to the organic compounds used in this work were also determined.     

Iron deposition in apoferritin. Evidence for the formation of a mixed valence binuclear iron complex

A preliminary EPR investigation of iron accumulation in apoferritin has identified paramagnetic species generated during the early stage of iron deposition within the apoprotein shell. A featureless resonance at g' = 4.3, attributable to solitary high spin Fe3+ ions bound to the protein, is generated when Fe(II) is added to apoferritin at a level of 0.5 Fe/subunit (12 Fe/molecule) followed by air oxidation. This resonance accounts for 36% of the added iron. The remainder is EPR-silent and is probably present as oligomeric Fe3+ species. The intensity of the g' = 4.3 signal is reduced 3-fold upon anaerobic addition of 5 Fe(II)/subunit as a new iron resonance with g' values of 1.94, 1.87, and 1.80 is generated. This signal is observable only at temperatures near that of liquid helium and resists saturation at power levels of 100 milliwatts. Its distinctive g-factors, temperature dependence, and saturation characteristics suggest that it arises from a spin-coupled Fe(II)-Fe(III) dimer having a net electron spin of 1/2. In accord with this idea, the signal disappears when air is admitted, presumably because of oxidation of the Fe(II). The proposed mixed valence dimer may be an important intermediate formed during the initiation of core formation within the protein shell.     

EPR spectra of photosystem I and other iron protein components in intact cells of cyanobacteria.

Electron paramagnetic resonance (EPR) spectra were recorded of whole filaments of the cyanobacteria Nostoc muscorum and Anabaena cylindrica. Signals due to manganese were removed by freezing and thawing the cells in EDTA. EPR spectra were assigned on the basis of their g values, linewidths, temperature dependence and response to dithionite and light treatments. The principal components identified were: (i) rhombic Fe3+ (signal at g = 4.3), probably a soluble storage form of iron; (ii) iron-sulfur centers A and B of Photosystem I; (iii) the photochemical electron acceptor 'X' of Photosystem I; this component was also observed for the first time in isolated heterocysts; (iv) soluble ferredoxin which was present at a concentration of 1 molecule per 140 +/- 20 chlorophyll molecules; (v) a membrane-bound iron-sulfur protein (g = 1.92). A signal g = 6 in the oxidized state was probably due to an unidentified heme compound. During deprivation of iron the rhombic Fe3+, centers A, B and X of Photosystem I, and soluble ferredoxin were all observed to decrease.     

Role of phosphate in initial iron deposition in apoferritin

Ferritins from microorganisms to man are known to contain varying amounts of phosphate which has a pronounced effect on the structural and magnetic properties of their iron mineral cores. The present study was undertaken to gain insight into the role of phosphate in the early stages of iron accumulation by ferritin. The influence of phosphate on the initial deposition of iron in apoferritin (12 Fe/protein) was investigated by EPR, 57Fe M?ssbauer spectroscopy, and equilibrium dialysis. The results indicate that phosphate has a significant influence on iron deposition. The presence of 1 mM phosphate during reconstitution of ferritin from apoferritin, Fe(II), and O2 accelerates the rate of oxidation of the iron 2-fold at pH 7.5. In the presence or absence of phosphate, the rate of oxidation at 0 degrees C follows simple first-order kinetics with respect to Fe(II) with half-lives of 1.5 +/- 0.3 or 2.8 +/- 0.2 min, respectively, consistent with a single pathway for iron oxidation when low levels of iron are added to the apoprotein. This pathway may involve a protein ferroxidase site where phosphate may bind iron(II), shifting its redox potential to a more negative value and thus facilitating its oxidation. Following oxidation, an intermediate mononuclear Fe(III)-protein complex is formed which exhibits a transient EPR signal at g' = 4.3. Phosphate accelerates the rate of decay of the signal by a factor of 3-4, producing EPR-silent oligonuclear or polynuclear Fe(III) clusters. In 0.5 mM Pi, the signal decays according to a single phase first-order process with a half-life near 1 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

EPR characteristics of chloride-depleted photosystem II membranes in the presence of other anions.

Chloride is an essential cofactor for the oxidation of water to oxygen. Anion substitution (Br(-), I(-), NO(2)(-), F(-)) in Cl(-)-depleted PS II membranes brings out significant changes in the EPR signals arising from the S(2) state and from the iron-quinone complex of PS II. On the basis of the changes observed in the S(2) state multiline signal and the Q(A)Fe(3+) EPR signal in Cl(-)-depleted PS II membranes after substituting with various anions, we report a possible binding site of anions such as chloride and bromide at the PS II donor side as well as at the acceptor side.     

Novel trifunctional carrier molecule for the fluorescent labeling of haptens

We developed a novel trifunctional carrier molecule for the synthesis of hapten-fluorophore conjugates as reporter molecules in immunoassays. This carrier eliminates some of the disadvantages associated with currently used fluorophore-labeling procedures including high nonspecific binding. The backbone of the carrier consists of the 21 amino acid residues of the insulin A-chain molecule. This polypeptide provides a single site (terminal amino group) for covalent coupling of the hapten, three carboxyl groups for the attachment of fluorophores, and four sulfhydryl groups for derivatization with hydrophilic residues to compensate for the hydrophobic effect of the attached fluorophores. The sites for fluorophore attachment are 4, 17, and 21 amino acids away from the hapten attachment site. This spatial separation minimizes quenching of the fluorescence signal due to interaction of the fluorophores with each other and with the attached hapten. In this study, 2,4-dinitrophenol (DNP) was selected as model hapten, fluorescein as label, and S-sulfonate groups as hydrophilic residues. The properties of the DNP-insulin A-chain-fluorescein conjugate (DNP-Ins-Fl) were compared to those of a DNP derivative labeled with a single fluorescein moiety via a small lysine spacer (DNP-Lys-Fl). The DNP-Ins-Fl conjugate exhibited a 3-fold lower nonspecific adsorption to immobilized non-immune IgG contributing to an approximately 3-fold more efficient displacement from the binding sites of an immobilized monoclonal anti-DNP antibody by the antigen DNP-lysine. Furthermore, at equimolar concentrations the DNP-Ins-Fl generated a 2.6-fold higher fluorescent signal than DNP-Lys-Fl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Redox properties and activity studies on a nickel-containing hydrogenase isolated from a halophilic sulfate reducer Desulfovibrio salexigens

A soluble hydrogenase from the halophilic sulfate reducing bacterium Desulfovibrio salexigens, strain British Guiana (NCIB 8403) has been purified to apparent homogeneity with a final specific activity of 760 mumoles H2 evolved/min/mg (an overall 180-fold purification with 20% recovery yield). The enzyme is composed of two non-identical subunits of molecular masses 62 and 36 kDa, respectively, and contains approximately 1 Ni, 12-15 Fe and 1 Se atoms/mole. The hydrogenase shows a visible absorption spectrum typical of an iron-sulfur containing protein (A400/A280 = 0.275) and a molar absorbance of 54 mM-1cm-1 at 400 nm. In the native state (as isolated, under aerobic conditions), the enzyme is almost EPR silent at 100 K and below. However, upon reduction under H2 atmosphere a rhombic EPR signal develops at g-values 2.22, 2.16 and around 2.0, which is optimally detected at 40 K. This EPR signal is reminiscent of the nickel signal C (g-values 2.19, 2.16 and 2.02) observed in intermediate redox states of the well characterized D. gigas nickel containing hydrogenase and assigned to nickel by 61 Ni isotopic substitution (J.J.G. Moura, M. Teixeira, I. Moura, A.V. Xavier and J. Le Gall (1984), J. Mol. Cat., 23, 305-314). Upon longer incubation with H2 the "2.22" EPR signal decreases. During the course of a redox titration under H2, this EPR signal attains a maximal intensity around--380 mV. At redox states where this "2.22" signal develops (or at lower redox potentials), low temperature studies (below 10 K) reveals the presence of other EPR species with g-values at 2.23, 2.21, 2.14 with broad components at higher fields. This new signal (fast relaxing) exhibits a different microwave power dependence from that of the "2.22" signal, which readily saturates with microwave power (slow relaxing). Also at low temperature (8 K) typical reduced iron-sulfur EPR signals are concomitantly observed with gmed approximately 1.94. The catalytic properties of the enzyme were also followed by substrate isotopic exchange D2/H+ and H2 production measurements.     

The Fe-only nitrogenase and the Mo nitrogenase from Rhodobacter capsulatus: a comparative study on the redox properties of the metal clusters present in the dinitrogenase components.

The dinitrogenase component proteins of the conventional Mo nitrogenase (MoFe protein) and of the alternative Fe-only nitrogenase (FeFe protein) were both isolated and purified from Rhodobacter capsulatus, redox-titrated according to the same procedures and subjected to an EPR spectroscopic comparison. In the course of an oxidative titration of the MoFe protein (Rc1Mo) three significant S = 1/2 EPR signals deriving from oxidized states of the P-cluster were detected: (1) a rhombic signal (g = 2.07, 1.96 and 1.83), which showed a bell-shaped redox curve with midpoint potentials (Em) of -195 mV (appearance) and -30 mV (disappearance), (2) an axial signal (g(parallel) = 2.00, g perpendicular = 1.90) with almost identical redox properties and (3) a second rhombic signal (g = 2.03, 2.00, 1.90) at higher redox potentials (> 100 mV). While the 'low-potential' rhombic signal and the axial signal have been both attributed to the one-electron-oxidized P-cluster (P1+) present in two conformationally different proteins, the 'high-potential' rhombic signal has been suggested rather to derive from the P3+ state. Upon oxidation, the FeFe protein (Rc1Fe) exhibited three significant S = 1/2 EPR signals as well. However, the Rc1Fe signals strongly deviated from the MoFe protein signals, suggesting that they cannot simply be assigned to different P-cluster states. (a) The most prominent feature is an unusually broad signal at g = 2.27 and 2.06, which proved to be fully reversible and to correlate with catalytic activity. The cluster giving rise to this signal appears to be involved in the transfer of two electrons. The midpoint potentials determined were: -80 mV (appearance) and 70 mV (disappearance). (b) Under weakly acidic conditions (pH 6.4) a slightly altered EPR signal occurred. It was characterized by a shift of the g values to 2.22 and 2.05 and by the appearance of an additional negative absorption-shaped peak at g = 1.86. (c) A very narrow rhombic EPR signal at g = 2.00, 1.98 and 1.96 appeared at positive redox potentials (Em = 80 mV, intensity maximum at 160 mV). Another novel S = 1/2 signal at g = 1.96, 1.92 and 1.77 was observed on further, enzymatic reduction of the dithionite-reduced state of Rc1Fe with the dinitrogenase reductase component (Rc2Fe) of the same enzyme system (turnover conditions in the presence of N2 and ATP). When the Rc1Mo protein was treated analogously, neither this 'turnover signal' nor any other S = 1/2 signal were detectable. All Rc1Fe-specific EPR signals detected are discussed and tentatively assigned with special consideration of the reference spectra obtained from Rc1Mo preparations.     

On the novel H2-activating iron-sulfur center of the "Fe-only" hydrogenases

The two hydrogenases (I and II) of the anaerobic N2-fixing bacterium Clostridium pasteurianum (Cp) and the hydrogenases of the anaerobes Megasphaera elsdenii (Me) and Desulfovibrio vulgaris (strain Hildenborough, Dv), contain iron-sulfur clusters but not nickel. They are the most active hydrogenases known. All four enzymes in their reduced states give rise to EPR signals typical of [4Fe-4S]1+ clusters but exhibit novel EPR signals in their oxidized states. For example, Cp hydrogenase I exhibits a sharp rhombic EPR signal when oxidized under mild conditions but the enzyme is inactivated by over-oxidation and then exhibits an axial EPR signal. A similar axial signal is observed from mildly oxidized hydrogenase I after treatment with CO. EPR, M?ssbauer and ENDOR spectroscopy indicate that the EPR signals from the oxidized enzyme and its CO derivative arise from a novel spin-coupled Fe center. Low temperature magnetic circular dichroism (MCD) studies reveal that an EPR-silent Fe-S cluster with S greater than 1/2 is also present in oxidized hydrogenase I. From a study of all spectroscopic properties of Cp, Dv, and Me hydrogenases, it is concluded that the H2-activating site of all four is a novel Fe-S cluster with S greater than 0 and integer, which in the oxidized state is exchange-coupled to a S = 1/2 species. The data are most consistent with the S = 1/2 species being a low spin Fe(III) center. The H2-activating site is susceptible to oxidative rearrangements to yield both active and inactive states of the enzyme. We discuss the possible implications of these finding to methods of enzyme oxidation and purification procedures currently used for hydrogenases.     

An EPR study of the pH dependence of formate effects on Photosystem II.

Effects of formate on rates of O(2) evolution and electron paramagnetic resonance (EPR) signals were observed in the oxygen evolving PS II membranes as a function of pH. In formate treated PS II membranes, decrease in pH value resulted in the inhibition of the O(2) evolving activity, a decrease in the intensity of S(2) state multiline signal but an increase in the intensity of the Q(A)(-)Fe(2+) EPR signal. Time-resolved EPR study of the Y(Z)(*) decay kinetics showed that the light-induced intensity of Y(Z)(*) EPR signal was proportional to the formate concentration. The change in the pH affected both the light-induced intensities and the decay rates of Y(Z)(*), which was found to be faster at lower pH. At 253 K, t(1/e) value of Y(Z)(*) decay kinetics was found to be 8-10 s at pH 6.0 and 18-21 s at pH 5.0. The results presented here indicate that the extent of inhibition at the donor and the acceptor side of PS II due to formate is pH dependent, being more effective at lower pH.     

Preparation and spectroscopic characterization of a coupled binuclear center in cobalt(II)-substituted hemocyanin.

A binuclear cobalt derivative of arthropod hemocyanin (Hc) has been prepared by the reaction of apo-Hc with Co(II) in the presence of thiocyanate. The crude product of the reaction contains specifically and adventitiously bound metal, the latter being removable by EDTA treatment. The specifically bound Co(II) constitutes a binuclear metal center that exhibits optical and CD spectra typical in their absorption maxima and extinction coefficients of Co(II) complexes with near-tetrahedral geometry. The EPR spectrum of the binuclear Co(II) derivative contains a resonance at g approximately 13, which is characteristic of integer spin systems and indicates coupled metal ions; the excess Co(II) bound to crude products exhibits an EPR signal at g approximately 4. The time course of derivative formation was followed by EPR, optical and atomic absorption techniques, and by fluorimetry. The intensity of the optical absorption in the visible region due to Co(II) increases with increasing stoichiometry of specifically bound metal [up to 2 Co(II) per protein monomer], but the intensity of the Co(II) EPR signal increases only during the formation of a mononuclear derivative. As the reaction proceeds over approximately 100 h to the formation of the binuclear derivative, the EPR signal intensity decreases to 10% of the value expected for 2 mol of EPR-active Co(II)/mol of protein. The binuclear cobalt derivative cannot be reconstituted to native Hc with Cu(I), indicating the stable loading of Co(II) in the active site. EPR and optical spectroscopic evidence is presented showing that the binuclear derivative does not bind oxygen.     

Characterization of the cycle of iron-mediated electron transfer from Adriamycin to molecular oxygen

The anticancer drug adriamycin binds iron and these complexes cycle to reduce molecular oxygen (Zweier, J. L. (1984) J. Biol. Chem. 259, 6056-6058). Optical absorption, EPR, and M?ssbauer spectroscopic data are correlated with polarographic O2 consumption and chemical Fe2+ extraction measurements in order to characterize each step in this cycle. Fe3+ binds to adriamycin at physiologic pH forming a complex with an optical absorbance maximum at 600 nm. EPR signals at g = 4.2 and g = 2.01, and a doublet M?ssbauer spectrum with isomer shift delta = 0.57 mm/s and quadrupole splitting delta EQ = 0.74 mm/s are observed indicating that the Fe3+ bound to adriamycin is high spin S = 5/2. Under anaerobic conditions the absorbance maximum at 600 nm decreases with an exponential decay constant = 0.77 h-1, and the EPR and M?ssbauer spectra of Fe3+-adriamycin similarly decrease as the Fe3+ is reduced to EPR silent Fe2+. The Fe2+-adriamycin complex which is formed exhibits a M?ssbauer spectrum with delta = 1.18 mm/s and delta EQ = 1.82 mm/s indicative of high spin Fe2+. As the EPR spectra of Fe3+-adriamycin decrease on reduction of the Fe3+ to Fe2+ a signal of the oxidized adriamycin free radical appears at g = 2.004 with line width of 8 G. On exposure to O2 the absorption maximum at 600 nm, the Fe3+ EPR, and the Fe3+ M?ssbauer spectra all return. Polarographic measurements demonstrate that O2 is consumed and that H2O2 is formed. Addition of high affinity Fe2+ chelators block O2 consumption indicating that Fe2+ formation is essential for O2 reduction. This cycle of iron-mediated O2 reduction can explain the formation of the reactive reduced oxygen and adriamycin radicals which are thought to mediate the biological activity of adriamycin.