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H Beinert

Publications and source records attributed to H Beinert.

At least 91 records · Page 5Linked to original sources
PubMed1982

Mössbauer studies of beef heart aconitase: evidence for facile interconversions of iron-sulfur clusters.

Beef heart aconitase, isolated under aerobic conditions, has been studied with Mössbauer and EPR spectroscopy. In the oxidized state, the enzyme exhibits an EPR signal at g = 2.01. The Mössbauer data show that this signal is associated with a 3Fe cluster. In dithionite-reduced aconitase, the 3Fe cluster, probably of the [3Fe-3S] type, is in a paramagnetic state of interger electronic spin (S = 2); the Mössbauer spectra exhibit al the unique features reported for proteins with 3Fe clusters. On activation of aconitase with ferrous ion, the paramagnetic 3Fe cluster of dithionite-reduced enzyme is converted into a diamagnetic (S = 0) form. Activation studies with iron enriched in either 27 Fe or 56 Fe suggest that activation transforms the 3Fe cluster into a center that has a [4Fe-4S] core. This conclusion is supported by the observation that EPR signals characteristic of reduced [4Fe-4S] clusters can be elicited under appropriate conditions. It has frequently been assumed that the activation of aconitase with Fe2+ produces an active site containing a single ferrous ion. The data reported here suggest that a ferrous ion is used to rebuild a [4Fe-4S] cluster.

Aconitate Hydratase↗
PubMed1982

Mitochondrial NADH dehydrogenase in iron-deficient and iron-repleted rat muscle: an EPR and work performance study.

Iron may affect both respiratory O2 transport and mitochondrial electron transport in the performance of muscle work. This study was designed to elucidate the molecular defect of iron-deficient work performance by identifying heretofore unmeasurable mitochondrial enzymes that are diminished by iron deficiency and may be restored by iron repletion. Female rats were made iron-deficient by dietary control and were repleted by oral iron. Iron deficiency reduced physical work capacity (treadmill running time), haemoglobin (Hb), and mitochondrial iron-sulphur (Fe-S) centres in heart and skeletal muscles; mitochondrial number was unaffected. Oral iron supplementation restored work capacity and Hb within 4 d to normal or near-normal levels, but in general Fe-S centres of mitochondria due to NADH dehydrogenase remained at iron-deficient levels. Subnormal concentrations of mitochondrial iron-dependent NADH dehydrogenase in muscle are not by themselves rate-limiting in work performance.

Anemia, Hypochromic↗
PubMed1981

Relationship of the oxidation state of the iron-sulfur cluster of aconitase to activity and substrate binding.

It is known that aconitase from mammalian mitochondria is only partially active as isolated but may be activated by incubation with iron, ascorbate, and a thiol, or with dithionite. It has been suggested that the added Fe in the activation mixture is essential for activation and that it is incorporated in the enzyme [Villafranca, J. J., & Mildvan, A. S. (1971) J. Biol. Chem. 246, 772-779; Gawron, O., Waheed, A., Glaid, A. J., & Jaklitsch, A. (1974) Biochem. J. 139, 709-714]. However, it is shown in this paper that, when the enzyme has a full complement of 3Fe and 3S, full activation is reached coulometrically, without iron or other chemical reducing agents. It is clear, therefore, that the role of activators is to reduce the iron--sulfur cluster of the enzyme. The appearance of catalytic activity on reduction of the cluster shows a pronounced lag, as does the decay of activity after reoxidizing the cluster. This suggests that catalytic activity requires a conformational change in the protein which is initiated by reduction of the cluster and that, following reoxidation, activity disappears only after the inactive conformation is assumed. Citrate and the competitive inhibitor trans-aconitate are bound to a comparable extent to the active and inactive forms, but only the active form can bind 1-hydroxy-2-nitro-1,3-propanedicarboxylic acid, a transition-state analogue. This is interpreted to show that in the inactive state aconitase cannot enter the conformation it assumes in the transition state during catalysis.

Aconitate Hydratase↗
PubMed1981

Oxidation of reduced cytochrome c oxidase with 18O2. A search for mu-oxo-bridged metal species in the oxidized enzyme.

We have measured the increase in 18O content of water produced from single turnover oxidations of anerobically reduced cytochrome c oxidase with 18O2 in order to test the hypothesis that a reduced atom of oxygen, originating from dioxygen, remains bound to oxidized cytochrome c oxidase in the form of a mu-oxo-bridge between two metal components when a single turnover occurs. When water samples produced by oxidizing the reduced enzyme with 18O2 were compared to natural abundance control samples obtained by oxidizing with 16O2, all of the 18O2 reduced in a single turnover could be accounted for in the form of additional H218O produced. We conclude that neither atom of the dioxygen reduced is incorporated into the enzyme as a bridge which is stable in the absence of oxidoreductive reactions on the time scale of several minutes.

Anaerobiosis↗
PubMed1981

Extended x-ray absorption fine structure of copper in cytochrome c oxidase: Direct evidence for copper-sulfur ligation.

The copper x-ray fluorescence excitation spectrum of cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) has been recorded in the 245-270 K range. The beat pattern observed in the extended x-ray absorption fine structure can be accounted for only by postulating a combination of sulfur and nitrogen (or oxygen) ligands to the copper. The average Cu-S distance is 2.27 +/- 0.02 A and the average Cu-N (or Cu-O) distance is 1.97 +/- 0.02 A. The amplitudes require ca, 1-1.5 sulfurs and 2 nitrogens (or oxygens) per copper. The distribution of sulfur ligands between Cu(A) and Cu(B) sites is not known, although there is some evidence that two sulfur atoms are bound to Cu(A).

Journal Article↗
PubMed1980

Electron spin echo studies of cytochrome c oxidase.

We have studied the linear electric field effect in pulsed EPR of the "EPR-detectable copper" signal of beef heart cytochrome c oxidase and have compared our results with those for a variety of square planar and tetrahedral Cu(II) model compounds and with Cu(II) proteins containing either type 1 or type 2 copper. The electric field induced g shifts (linear electric field effect) for cytochrome oxidase are comparable in magnitude to those for simple Cu(II) complexes and for some copper proteins containing type 2 sites. The shifts are smaller than those for tetrahedral copper complexes and for type 1 copper sites. However, the magnetic field dependence of the linear electric field effect does not resemble that observed for any Cu(II) complex studied nor for type 1 copper. These findings cannot be reconciled with the tetrahedral Cu(II) model proposed by Greenaway, Chan, and Vincow ((1977) Biochim. Biophys. Acta 490, 62-78, 1977) to explain the unusual EPR spectrum of cytochrome oxidase.

Animals↗
PubMed1980

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.

Animals↗
PubMed1979

The oxygen reactions of reduced cytochrome c oxidase. Position of a form with an unusual EPR signal in the sequence of early intermediates.

The order of appearance of intermediates in the reoxidation of reduced cytochrome c oxidase by oxygen has been examined. Particular emphasis was placed on determining where the intermediate with the EPR signal at g = 5, 1.78, 1.69 (Shaw, R.W., Hansen, R.E. and Beinert, H. (1978) J. Biol. Chem. 253, 6637--6640) appears in the sequence of events during reoxidation. Flash photolysis of reduced, CO-complexed samples of cytochrome c oxidase in the presence of oxygen in a buffer containing 30% (v/v) ethylene glycol at 77 K and 195 K has been used to generate states of partial reoxidation. The intermediate with the EPR signal at g = 5, 1.78, and 1.69 can be detected as a product of the photolysis and subsequent oxidation but does not appear until the photolyzed sample is incubated at temperatures well above 196 K. In the course of the reoxidation, the intermediate characterized by the g = 5, 1.78, 1.69 signal occurs in the reaction sequence after the states referred to as 'Compound A' and 'Compound B' (Chance, B., Saronio, C., and Leigh, J.S. (1975) J. Biol. Chem. 250, 9226--9237). Its appearance is within the time range reported for the formation of 'oxygenated' cytochrome c oxidase (Orii, Y. (1979) in Cytochrome Oxidase (King, T.E., Orii, Y., Chance, B. and Okunuki, K., eds.), pp. 331--340, Elsevier/North-Holland Biomedical Press, Amsterdam).

Animals↗
PubMed1979

The high potential iron-sulfur cluster of aconitase is a binuclear iron-sulfur cluster.

It has been reported (Ruzicka, F.J., and Beinert, H. (1978) J. Biol. Chem. 253, 2514-2517) that aconitase in the oxidized state, as isolated, shows an electron paramagnetic resonance signal centered at g = 2.01, typical of high potential iron-sulfur proteins. Since the magnetic state corresponding to this signal has thus far only been found in tetranuclear iron-sulfur clusters in model compounds and proteins, it could be expected that aconitase also contains a [4Fe-4S] cluster. We show here that core extrusion, in the presence of hexamethylphosphoramide and o-xylyl-alpha,alpha'-dithiol and subsequent ligand exchange with p-trifluoromethylbenzenethiol yield absorption spectra typical of binuclear iron-sulfur clusters. According to the absorbance measured, the concentration of the extruded [2Fe-2S] cluster quantitatively accounts for the iron-sulfur content of the preparations examined. Preliminary studies of the 19F nuclear magnetic resonance spectrum obtained on extrusion with p-trifluoromethylbenzenethiol confirm the presence of a binuclear cluster in aconitase.

Aconitate Hydratase↗
PubMed1978

The magnetic susceptibility of cytochrome oxidase in the 4.2-1.5 K range.

Sixteen low temperature measurements on eight independent cytochrome oxidase samples from two separate laboratories have yielded magnetic susceptibility data compatible with a model of spin-coupled iron and copper ions, as presented in the preceding paper (Tweedle, M.F., Wilson, L.J., García-Iñiguez, L., Babcock, G. T., and Palmer, G. (1978) J. Biol. Chem. 253, 8065-8071). The data in the 1.5-77 K range match those attained at higher temperatures and the predictions of the spin-coupled model. Measurements on reduced samples confirm the high spin nature of one iron atom. No obvious uncoupling of the antiferromagnetic Fe-Cu interaction is detected in partly reduced samples.

Copper↗
PubMed1978

Responses of the a3 component of cytochrome c oxidase to substrate and ligand addition.

We have previously described a transient high spin ferric heme species in cytochrome c oxidase (EC 1.9.3.1) which represent a3+(3) (Beinert, H. and Shaw, R.W.(1977) Biochim. Biophys. Acta 462, 12u--130), and can be detected and quantitatively determined by EPR. We have now used out ability to generate this species to study reactions of a3+(3) with substrates and ligands and also responses to pH changes. This was accomplished by multiple rapid mixing and freezing techniques in conjunction with low temperature EPR and optical reflectance spectroscopies. The substrates used were O2 and ferrocytochrome c and the ligands cyanide, sulfide, azide and carbon monoxide. Contrary to the oxidized, resting form of the enzyme, the transient high spin species of a3+(3) reacts within less than 10 ms stoichiometrically with cyanide and sulfide and at a slower rate with azide. The transient a3+(3) species responds to O2 and CO by changes in signal size or shape, although no oxidoreduction is involved, indicating that a3+(3) registers the presence of these gases. The high spin signal of the transient species is readily abolished by ferrocytochrome c or on raising the pH. Decreasing the pH induces a shift from the rhombic towards the axial component of the signal. Since the responses to CO and pH are analogous for the rhombic transient species to those observed with the rhombic high spin ferric heme species produced on partial reduction, it is suggested that the rhombic signals represent a3+(3) in either case. In all these experiments, in which EPR detectable a3+(3) was observed in large yield, no extra signals for copper or correspondingly increased intensity in the copper signal at g = 2 were seen. The relationship is discussed of the obviously reactive transient species of a3+(3) to other 'activated' species that have been reported and to the oxidized resting form of the enzyme, which is known to react only slowly with ligands and to respond sluggishly to substrate.

Azides↗
PubMed1978

A novel electron paramagnetic resonance signal of "oxygenated" cytochrome c oxidase.

It had been observed previously that a pair of transient EPR resonances (g = 1.78 and 1.69) appears within less than 5 ms on reoxidation of reduced cytochrome c oxidase by O2. Since the location of other lines that are part of the same signal was not known, the quantity of the paramagnetic species involved, and thus the significance of the observed resonances, remained questionable. We have now found a broad resonance at g = 5 which is obviously associated with those at g = 1.78 and 1.69. The width of the signal (approximately 250 mT) at the observed intensity suggests that it represents a significant fraction of one of the components of the enzyme. The signal disappears within less than 5 ms on addition of cyanide or sulfide but only within several hundred milliseconds after addition of ferrocytochrome c. This behavior suggests that it originates from the a3 component of the enzyme. It is suggested that the species represented in the signal is either identical with or part of what has been named collectively the "oxygenated" form and recently described "activated" forms of the enzyme. On reoxidation of reduced oxidase with oxygen enriched 90% in 17O, no change of signal shape was seen.

Cyanides↗
PubMed1978

The soluble "high potential" type iron-sulfur protein from mitochondria is aconitase.

Properties of soluble high potential type iron-sulfur protein (HiPIP) from beef heart mitochondria were compared to those of aconitase from pig heart. The two proteins when purified to homogeneity by the criteria of sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis show identical light absorption characteristics. EPR signals of the HiPIP type centered at g = 2.01 when oxidized, isoelectric points at pH 8.5 to 8.6, are inseparable by SDS-polyacrylamide electrophoresis, and exhibit aconitase activity when activated by reducing agents in the presence of ferrous iron. The requirement for activation goes parallel to the intensity of the signal from the oxidized iron-sulfur cluster, i.e. the cluster is reduced in the active enzyme. We conclude that the soluble mitochondrial HiPIP is identical with aconitase. The relationships of iron to labile sulfide, molecular weight and unpaired spins in the EPR signal, and implications of our findings for the role of iron in aconitase are discussed.

Aconitate Hydratase↗