Characteristics of sulfhydryl groups of histone H3 from the calf thymus using mercury-containing nitroxyl radicals

The interaction between total histone and deoxyribonucleoprotein (DNP) preparations from calf thymus with mercury-containing nitroxyl radicals in low ionic strength solutions, 2 M NaCl and urea was investigated. It was found that the label is rapidly incorporated into the SH-groups of histone H3 to produce characteristic EPR signals. Titration of SH-groups within DNP demonstrated that in low ionic strength solutions only one SH-group (presumably, the SH-group of the cysteine residue in position 110) is accessible to the reagents. After dissociation by 2 M NaCl, two SH-groups become titrable; however, the EPR spectra point to differences in the conformational state of these two groups. In 4 M urea, these differences are compensated for by structural disintegration. The spin labels may be used for the analysis of SH-groups under different conditions and at different functional states of nucleoproteins.     

The mechanism of photodamage of eye structures. IV. Changes in the reactivity of crystalline SH-groups during UV irradiation

The kinetics of individual crystalline SH-group modification by DTNB were studied. According to the rates of their interaction with the modifier, the thiol groups in the native protein molecule can be classified as free, accessible, weakly modified and "masked" ones. Denaturation by the detergent (CTAB) caused an increase in the SH-group modification rate. In this case the SH-groups were modified as free and accessible ones. Illumination with UV-light resulted in a decrease in the number of SH-groups, opening of "masked" SH-groups in almost all crystallines except for alpha-crystalline, and essential changes in the SH-group modification rate.     

Characterization of two soluble ferredoxins as distinct from bound iron-sulfur proteins in the photosynthetic bacterium Rhodospirillum rubrum.

In an earlier investigation (Shanmugam, K. T., Buchanan, B. B., and Arnon, D. I. (1972) Biochim. Biophys. Acta 256, 477-486) the extraction of ferredoxin from Rhodospirillum rubrum cells with the aid of a detergent (Triton X-100) and acetone revealed the existence of two types of ferredoxin (I and II) and led to the conclusion that both are membrane-bound. In the present investigation, ferredoxin and acid-labile sulfur analyses of photosynthetic membranes (chromatophores) and soluble protein extracts of the photosynthetic bacteria R. rubrum and Rhodopseudomonas spheroides showed that ferredoxins I and II are primarily components of the soluble protein fraction. After their removal, washed R. rubrum chromatophores were found to contain a considerable amount of tightly bound iron-sulfur protein(s), as evidenced by acid-labile sulfur and electron paramagnetic resonance analyses. Thus, like all other photosynthetic cells examined to date, R. rubrum cells contain both soluble ferredoxins and iron-sulfur proteins tightly bound to photosynthetic membranes. The molecular weights of ferredoxins I and II from photosynthetically grown R. rubrum cells were found to be 8,800 and 14,500, respectively. Using these molecular weights, the molar extinction coefficients at 390 nm for ferredoxins I and II were determined to be 30.3 and 17.2 mM-1 CM-1, respectively. Ferredoxin I contains 8 non-heme iron and 8 acid-labile sulfur atoms per molecule; ferredoxin II contains 4 non-heme iron and 4 acid-labile sulfur atoms per molecule. Ferredoxin I was found only in photosynthetically grown cells whereas ferredoxin II was present in both light- and dark-grown cells. Ferredoxin II from both light- and dark-grown cells has the same molecular weight (14,500) and absorption spectrum and has 4 iron and 4 acid-labile sulfur atoms per molecule. Low temperature electron paramagnetic resonance spectra of oxidized and photoreduced ferredoxins I and II from R. rubrum were recorded. The EPR spectrum of oxidized ferredoxin II exhibited a single resonance line at g = 2.012. Oxidized ferredoxin I, however, exhibited a spectrum that may arise from the superimposition of two resonance lines near g = 2.012. Photoreduced ferredoxin II displayed a rhombic EPR spectrum with a g value of 1.94. Photoreduced ferredoxin I exhibited a similar EPR spectrum at a temperature of 16 K, but when the temperature was lowered to 4.5 K the spectrum of ferredoxin I changed. This temperature-dependent spectrum may result from a weak spin-spin interaction between two iron-sulfur clusters. These results are consistent with the conclusion that R. rubrum ferredoxins I and II are, respectively, 8 iron/8 sulfur and 4 iron/4sulfur proteins.     

Isolation and characterization of a rubredoxin and two ferredoxins from Desulfovibrio africanus

Rubredoxin and two distinct ferredoxins have been purified from Desulfovibrio africanus. The rubredoxin has a molecular weight of 6000 while the ferredoxins appear to be dimers of identical subunits of approximately 6000 to 7000 molecular weight. Rubredoxin contains one iron atom, no acid-labile sulfide and four cysteine residues per molecule. Its absorbance ratio A₂₇₈/A₄₉₀ is 2.23 and its amino acid composition is characterized by the absence of leucine and a preponderance of acidic amino acids.

The two ferredoxins, designated I and II, are readily separated on DEAE-cellulose. The amino acid compositions of ferredoxins I and II show them to be different protein species; the greater number of acidic amino acid residues in ferredoxin I than in ferredoxin II appears to account for separation based on electronic charge. Both ferredoxins contain four iron atoms, four acid-labile sulfur groups and either four (ferredoxin II) or six (ferredoxin I) cysteine residues per molecule. Spectra of the two ferredoxins differ from those of ferredoxins of other Desulfovibrio species by exhibiting a pronounced absorption peak at 283 nm consistent with an unusual high content of aromatic residues. The A₃₈₅/A₂₈₃ absorbance ratio of ferredoxins I and II are 0.56 and 0.62, respectively.

The N-terminal sequencing data of the two ferredoxins clearly indicate that ferredoxins I and II are different protein species. However, the two proteins exhibit a high degree of homology.

The physiological activity of ferredoxins I and II appears to be similar as far as the electron transfer in the phosphoroclastic reaction is concerned.     

Isolation and characterization of a rubredoxin and two ferredoxins from Desulfovibrio africanus

Rubredoxin and two distinct ferredoxins have been purified from Desulfovibrio africanus. The rubredoxin has a molecular weight of 6000 while the ferredoxins appear to be dimers of identical subunits of approximately 6000 to 7000 molecular weight. Rubredoxin contains one iron atom, no acid-labile sulfide and four cysteine residues per molecule. Its absorbance ratio A278/A490 is 2.23 and its amino acid composition is characterized by the absence of leucine and a preponderance of acidic amino acids. The two ferredoxins, designated I and II, are readily separated on DEAE-cellulose. The amino acid compositions of ferredoxins I and II show them to be different protein species; the greater number of acidic amino acid residues in ferredoxin I than in ferredoxin II appears to account for separation based on electronic charge. Both ferredoxins contain four iron atoms, four acid-labile residues per molecule. Spectra of the two ferredoxins differ from those of ferredoxins of other Desulfovibrio species by exhibiting a pronounced absorption peak at 283 nm consistent with an unusual high content of aromatic residues. The A385/A283 absorbance ratio of ferredoxins I and II are 0.56 and 0.62, respectively. The N-terminal sequencing data of the two ferredoxins clearly indicate that ferredoxins I and II are different protein species. However, the two proteins exhibit a high degree of homology.     

Midpoint redox potentials of plant and algal ferredoxins.

Midpoint potentials of plant-type ferredoxins from a range of sources were measured by redox titrations combined with electron-paramagnetic-resonance spectroscopy. For ferredoxins from higher plants, green algae and most red algae, the midpoint potentials (at pH 8.0) were between --390 and --425 mV. Values for the major ferredoxin fractions from blue-green algae were less negative (between --325 and --390 mV). In addition, Spirulina maxima and Nostoc strain MAC contain second minor ferredoxin components with a different potential, --305 mV (the highest so far measured for a plant-algal ferrodoxin) for Spirulina ferrodoxin II, and --455 mV (the lowest so far measured for a plant-algal ferredoxin) for Nostoc strain MAC ferredoxin II. However, two ferredoxins extracted from a variety of the higher plant Pisum sativum (pea) had midpoint potentials that were only slightly different from each other. These values are discussed in terms of possible roles for the ferredoxins in addition to their involvement in photosynthetic electron transport.     

Study of reactivity of sulfhydryl groups of troponin]

The reactivities of SH-groups of troponin and its components were studied, using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). At low concentrations of Ca2+ (pCa greater than 8) one rapidly- and two slowly-reacting SH-groups of troponin I and one SH-group of troponin C slowly reacting with NBD-chloride are titrated in the whole troponin complex. When Ca2+ concentration is increased up to pCa less than 5, only one slowly-reacting and one rapidly-reacting with NBD-chloride SH-groups of troponin I are titrated. The increase of Ca2+ concentration from pCa greater than 8 to pCa less than 5 results in a change of the environment polarity for the highly reactive SH-group of troponin I in the whole troponin complex. This phenomenon may suggest that the changes in troponin C structure during Ca2+ binding somehow induce changes in that of troponin I. The half-maximal change of troponin SH-groups reactivity is found at pCa 6.8. No cooperativity for Ca2+ binding by the troponin complex is observed using SH-groups titration by NBD-Cl.     

Amino acid sequence of a four-iron-four-sulphur ferredoxin isolated from Bacillus stearothermophilus.

1. The primary structure of a 4Fe-4S ferredoxin from Bacillus stearothermophilus was determined and shown to consist of a single polypeptide chain of 81 amino acid residues. The molecular weight of the holoprotein is about 9120. 2. There are only four cysteine residues in the molecule; three of these are located near the N-terminus as a Cys-X-X-Cys-X-X-Cys segment, and the fourth cysteine residue is followed by a proline and located in the C-terminal half. 3. The Fe-S chromophore in B. stearothermophilus ferredoxin was previously well characterized and was shown to consist of a single 4Fe-4S cluster. This ferredoxin sequence establishes for the first time the relative location of the four cysteine residues necessary to bind the 4Fe-4S cluster of a 4Fe ferredoxin, and is in agreement with the criteria for the relative positions of the cysteines proposed from X-ray-crystallographic studies on an 8Fe (two 4Fe-4S clusters) ferredoxin. 4. The sequence of B. stearothermophilus ferredoxin is homologous in many segments to that of other bacterial ferredoxins, the degree of homology being greater towards ferredoxins from Desulfovibrio gigas and photosynthetic bacteria than to Clostridial ferredoxins. 5. The presence of a relatively higher number of glutamic acid and lower number of cysteine residues in the molecule may explain the greater thermal stability and oxygen-insenstivity of this ferredoxin.     

Physicochemical characterization of the four-iron-four-sulphide ferredoxin from Bacillus stearothermophilus.

1. A stable ferredoxin was prepared from Bacillus stearothermophilus and purified by chromatography on DEAE-cellulose and by electrophoresis. 2. The minimum molecular weight determined from the amino acid composition was about 7900 and this was in reasonable agreement with a value of 8500 determined by polyacrylamide-gel electrophoresis. The ferredoxin contained four iron atoms and four labile sulphide groups per molecule. 3. The optical absorption, optical-rotatory-dispersion and circular-dichroism spectra are typical of ferredoxins containing 4Fe-4S clusters. 4. Oxidation-reduction titrations, combined with electron-paramagnetic-resonance (e.p.r.) spectroscopy, showed that the protein has a mid-point potential, at pH8, of -280 +/- 10mV, and that only one electron-accepting paramagnetic species is present. 5. The e.p.r. spectrum of the reduced ferredoxin is more readily saturated with microwave power at low temperatures than those of the eight-iron ferredoxins, indicating that there is another mechanism of electron-spin relaxation in the latter. 6. Mossbauer spectra of both redox states were observed over a range of temperatures and in magnetic fields. At high temperatures (77 degrees K and above) both redox states appear as quadrupole-split doublets; in the reduced state two resolved doublets are seen, suggesting appreciable localization of the additional reducing electron. 7. The average chemical shift indicates formal valences of two Fe3+ and two Fe2+ in the oxidized state and three Fe2+ and one Fe3+ in the reduced state. However, the spectra indicate that there are differing degrees of electron delocalization over the iron atoms. 8. At low temperatures (4.2 degrees K) the oxidized form shows no hyperfine magnetic interaction, even in an applied magnetic field, evidence that the oxidized ferredoxin is in a non-magnetic state as a result of antiferromagnetic coupling between the iron atoms. 9. At 4.2 degrees K the reduced form shows a broad asymmetric pattern resulting from magnetic hyperfine interaction. This contrasts with the reduced ferredoxin of Clostridium pasteurianum, which shows a doublet, suggesting that in the latter there may be interaction between the two 4Fe-4S centres. 10. In large applied magnetic fields, positive and negative hyperfine fields are seen in the Mossbauer spectra of the reduced ferredoxin, evidence for antiferromagnetic coupling between the iron atoms in the 4Fe-4S centre. The high-field spectra of the reduced ferredoxin of B. stearothermophilus are similar to those of the reduced ferredoxin of C. pasteurianum.     

Both human ferredoxins 1 and 2 and ferredoxin reductase are important for iron-sulfur cluster biogenesis

Ferredoxins are iron-sulfur proteins that have been studied for decades because of their role in facilitating the monooxygenase reactions catalyzed by p450 enzymes. More recently, studies in bacteria and yeast have demonstrated important roles for ferredoxin and ferredoxin reductase in iron-sulfur cluster assembly. The human genome contains two homologous ferredoxins, ferredoxin 1 (FDX1) and ferredoxin 2 (FDX2--formerly known as ferredoxin 1L). More recently, the roles of these two human ferredoxins in iron-sulfur cluster assembly were assessed, and it was concluded that FDX1 was important solely for its interaction with p450 enzymes to synthesize mitochondrial steroid precursors, whereas FDX2 was used for synthesis of iron-sulfur clusters, but not steroidogenesis. To further assess the role of the FDX-FDXR system in mammalian iron-sulfur cluster biogenesis, we performed siRNA studies on FDX1 and FDX2, on several human cell lines, using oligonucleotides identical to those previously used, along with new oligonucleotides that specifically targeted each gene. We concluded that both FDX1 and FDX2 were important in iron-sulfur cluster biogenesis. Loss of FDX1 activity disrupted activity of iron-sulfur cluster enzymes and cellular iron homeostasis, causing mitochondrial iron overload and cytosolic iron depletion. Moreover, knockdown of the sole human ferredoxin reductase, FDXR, diminished iron-sulfur cluster assembly and caused mitochondrial iron overload in conjunction with cytosolic depletion. Our studies suggest that interference with any of the three related genes, FDX1, FDX2 or FDXR, disrupts iron-sulfur cluster assembly and maintenance of normal cytosolic and mitochondrial iron homeostasis.     

X-ray inactivation studies of nonheme iron proteins the action of X-rays on Spinach ferredoxin in aqueous solutions

Summary Exposure of aqueous spinach ferredoxin solutions to X-rays results in a rapid and irreversible denaturation of the molecule. The denaturation is manifested by a decrease of the characteristic absorption of spinach ferredoxin at 320 and 416 nm, and by the concomitant liberation of ferric iron and hydrogen sulfide. The absorption decrease at 320 and 416 nm and the iron liberation are found to parallel the activity decrease in functioning as electron transfer factor in the noncyclic electron transport system in spinach chloroplasts.X-ray inactivated spinach ferredoxin does not contain iron or free SH-groups, and can be regarded as anapo-form of the native protein. This X-ray-inactivated apoprotein, however, showed a higher molar extinction coefficient at 275 nm than the apoferredoxin, and was not reconstitutable.Spinach ferredoxin was found to be even more radiosensitive than clostridial ferredoxin. AG- value of 1.25 for biological inactivation and iron liberation was found, as compared to aG- value of 0.8 for clostridial ferredoxin.     

Synchronous conformational oscillations in the titer of sulfhydryl groups in protein solutions. Reversible oxidation as a possible cause of this phenomenon

In solutions of creatine kinase, actin and lactic dehydrogenase oscillations of SH-groups are revealed by three independent methods using three different reagents AgNO3, PCMB, DTNB. The variations of the SH-group titre remain after denaturation of the individual protein solution portions. In the solutions of predenaturated proteins no titre oscillations are observed. Possible cause of the observed oscillations of SH-groups titre may be reversible SH--SS transformation.     

Characterization of fumarate reductase from baker's yeast: essential sulfhydryl group for binding of FAD

Fumarate reductase apoenzyme having the ability to reconstitute active enzyme was obtained by dialyzing the holoenzyme against 1 M KBr. The dissociation constant of the FAD-apoenzyme complex was 2.3 X 10(-8) M. The denatured holoenzyme and apoenzyme possessed seven sulfhydryl (SH) groups as determined with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). In the native apoenzyme, five SH-groups reacted with DTNB, and four of them were completely protected by the addition of FAD, while in the native holoenzyme, one was modified without inactivation. These results indicate that one SH-group is located on the surface of the enzyme molecule, four at or near the FAD-binding site, and two deeply embedded in the molecule. The modification of the apoenzyme caused inhibition of binding of FAD, resulting in loss of the ability to reconstitute enzymatic activity. Analyses of the data by statistical and kinetic methods suggested that a reactive SH-group is involved among the four SH-groups in the binding of FAD to the apoenzyme.     

Electron transfer mechanism and interaction studies between cytochrome C3 and ferredoxin

Ferredoxin, cytochrome c3 and hydrogenase are specific partners of the sulfate reduction pathway of Desulfovibrio desulfuricans Norway and might be exemplary for electron exchange mechanism studies. Cytochrome c3 contains four low redox potential haems for 13 000 molecular weight. Two ferredoxins isolated from the same bacteria are dimers of 6 000 molecular weight per subunit (Ferredoxin I: one (4 Fe-4S) cluster per subunit, ferredoxin II: two (4 Fe-4 S) clusters per subunit). The amino acid sequence of ferredoxin I is reported and compared to the ferredoxin II sequence. The structural characteristics of ferredoxins and cytochrome c3 should allow a discussion on the nature of the interaction. 1H-NMR spectra of ferredoxin I and cytochrome c3 in the absence and presence of ferredoxin are presented.     

Laser flash photolysis studies of the kinetics of reduction of spinach and Clostridium ferredoxins by a viologen analogue: electrostatically controlled nonproductive complex formation and differential reactivity among the iron-sulfur clusters

We have studied the transient kinetics of electron transfer from a positively charged viologen analogue (propylene diquat), reduced by pulsed laser excitation of the deazariboflavin/EDTA system, to the net negatively charged ferredoxins from spinach and Clostridium pasteurianum. Spinach ferredoxin showed monophasic kinetics over the ionic strength range studied, consistent with the presence of only a single iron-sulfur center. Clostridium ferredoxin at low ionic strength showed biphasic kinetics, which indicates a differential reactivity of the two iron-sulfur centers of this molecule toward the electron donor. The kobsd values for the initial fast phase observed with Clostridium ferredoxin were ionic strength dependent, whereas the slow-phase kinetics were ionic strength independent. This correlates with the highly asymmetric charge distribution on the surface of the bacterial protein relative to the two iron-sulfur clusters. The kinetics corresponding to spinach ferredoxin reduction were also ionic strength dependent, and the results obtained with these kinetics and with the fast phase of the bacterial ferredoxin reduction were consistent with a mechanism involving electrostatically stabilized complex formation. For spinach ferredoxin, the second-order rate constant extrapolated to infinite ionic strength was 2-fold smaller, and the extrapolated limiting first-order rate constant was 10-fold smaller, than for Clostridium ferredoxin, indicating a smaller intrinsic reactivity of the spinach protein toward the electron donor. Differences in the rate constant values and the ionic strength dependencies with both ferredoxins are consistent with differences in cluster structure and environment and protein size and charge distribution. For both proteins, the total amount of ferredoxin reduced increased with the ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of oligomerization on the properties of essential SH-groups of mitochondrial creatine kinase

The properties of creatine kinase isolated from bovine heart mitochondria in dimeric (Mr = 84 +/- 6 kD) and octameric (Mr = 340 +/- 17 kD) forms were compared with those of the earlier described hexameric form of the enzyme (Mr = 240 +/- 12 kD). The kinetics of SH-group modification by DTNB, the inactivation kinetics as well as the number of modified SH-groups point to significant differences between the three oligomeric forms of the enzyme. Each subunit of creatine kinase was found to possess one "fast" essential cysteine residue whose modification by DTNB and iodoacetamide led to enzyme inactivation. The formation of an analog of the transition state complex (E--MgADP--NO3--creatine) was paralleled with partial protection of only the "fast" cysteine residue which manifested itself in the decrease of the rate of its interaction with DTNB in all the three oligomeric forms. Dimer association into a hexamer and octamer occurred in parallel with a decrease of the affinity of essential SH-groups of cysteine for DTNB in 50% of the oligomeric molecule subunits. Thus, in the dimer two essential SH-groups were rapidly modified by DTNB at the same rate: k1 = k2 = (23.9 +/- 5.6).10(4) M-1 min-1. Within the hexamer, the rate of modification of 3 out of 6 SH-groups was practically unchanged: k1 = (10.6 +/- 2.3).10(4) M-1 min-1. Another 3 SH-groups in the remaining 50% of the subunits were partly masked, which manifested itself in a 10-fold decrease of their modification rate: k2 = (1.12 +/- 0.28).10(4) M-1 min-1. Within the octamer, the SH-groups rapidly interacted with DTNB only on 4 subunits: k1 = (20.7 +/- 2.2).10(4) M-1 min-1, whereas in the remaining 4 octamer subunits a practically complete masking of essential SH-groups was observed, as a result of which these groups became inaccessible to DTNB. This manifested itself in a 1000-fold decrease of the rate of SH-group modification by DTNB which reached that of non-essential SH-group modification. In has been found that a complete loss of the octamer activity is due to the modification of only 4 SH-groups which interact with DTNB at a high rate. A model for subunit association into a dimer, hexamer and octamer has been proposed. Presumably, 50% of the active centers in the mitochondrial creatine kinase octamer are not involved in the catalytic act.     

The protein responsible for center A/B in spinach photosystem I: isolation with iron-sulfur cluster(s) and complete sequence analysis

The 9 kDa polypeptide from spinach photosystem I (PS I) complex was isolated with iron-sulfur cluster(s) by an n-butanol extraction procedure under anaerobic conditions. The polypeptide was soluble in a saline solution and contained non-heme irons and inorganic sulfides. The absorption spectrum of this iron-sulfur protein was very similar to those of bacterial-type ferredoxins. The amino acid sequence of the polypeptide was determined by using a combination of gas-phase sequencer and conventional procedures. It was composed of 80 amino acid residues giving a molecular weight of 8,894, excluding iron and sulfur atoms. The sequence showed the typical distribution of cysteine residues found in bacterial-type ferredoxins and was highly homologous (91% homology) to that deduced from the chloroplast gene, frxA, of liverwort, Marchantia polymorpha. The 9 kDa polypeptide is considered to be the iron-sulfur protein responsible for the electron transfer reaction in PS I from center X to [2Fe-2S] ferredoxin, namely a polypeptide with center(s) A and/or B in PS I complex. It is noteworthy that the 9 kDa polypeptide was rather hydrophilic and a little basic in terms of the primary structure. A three-dimensional structure was simulated on the basis of the tertiary structure of Peptococcus aerogenes [8Fe-8S] ferredoxin, and the portions in the molecule probably involved in contacting membranes or other polypeptides were indicated. The phylogenetic implications of the structure of the present polypeptide as compared with those of several bacterial-type ferredoxins are discussed.     

Phosphate carrier of liver mitochondria: two equivalent SH-groups in the carrier unit

Phosphate carrier activity of mitochondria that had become swollen during the aerobic accumulation of calcium acetate was measured indirectly by monitoring the turbidity changes. The exchange between extramitochondrial phosphate and intramitochondrial acetate was inhibited by SH-group binding reagents. Part of the SH-groups of the carrier could be blocked by mersalyl without loss of activity whereas liberation of the same groups restored carrier activity when all the other SH-groups were irreversibly blocked. A symmetrical structure of the carrier is proposed with two equivalent SH-groups; each of them is able in itself to maintain carrier function.     

Crystal structure determination at 1.4 A resolution of ferredoxin from the green alga Chlorella fusca.

BACKGROUND: [2Fe-2S] ferredoxins, also called plant-type ferredoxins, are low-potential redox proteins that are widely distributed in biological systems. In photosynthesis, the plant-type ferredoxins function as the central molecule for distributing electrons from the photolysis of water to a number of ferredox-independent enzymes, as well as to cyclic photophosphorylation electron transfer. This paper reports only the second structure of a [2Fe-2S] ferredoxin from a eukaryotic organism in its native form. RESULTS: Ferredoxin from the green algae Chlorella fusca has been purified, characterised, crystallised and its structure determined to 1.4 A resolution - the highest resolution structure published to date for a plant-type ferredoxin. The structure has the general features of the plant-type ferredoxins already described, with conformational differences corresponding to regions of higher mobility. Immunological data indicate that a serine residue within the protein is partially phosphorylated. A slightly electropositive shift in the measured redox potential value, -325 mV, is observed in comparison with other ferredoxins. CONCLUSIONS: This high-resolution structure provides a detailed picture of the hydrogen-bonding pattern around the [2Fe-2S] cluster of a plant-type ferredoxin; for the first time, it was possible to obtain reliable error estimates for the geometrical parameters. The presence of phosphoserine in the protein indicates a possible mechanism for the regulation of the distribution of reducing power from the photosynthetic electron-transfer chain.     

Aspartate aminotransferase from chicken heart cytosol. Characterization of SH-groups]

Homogeneous aspartate aminotransferase has been prepared from chicken heart cytosol. The purification procedure includes fractionation with NH4-sulfate and with ethanol, chromatography on ion-exchange cellulose DE-32 and on hydroxylapatite. Crystallization of the enyme is described. The enzyme was shown to contain 4 SH-groups per protein subunit of molecular weight 50 000. Two of the SH-groups are fully buried, they can be blocked with thiol reagents only upon denaturation of the protein. One exposed SH-group is readily modified at alkaline pH by iodoacetamide, N-ethymaleimide or tetranitromethane, without any inhibition of enzymic activity; this group readily reacts also with 5,5,-ditthiobis (2-nitrobenzoate) and p-mercuribenzoate. One SH-group is semi-buried: it is inaccessible to the above-mentioned reagents at pH 8, but can be blocked by p-mercuribenzoate at pH about 5. Blocking with p-mercuribenzoate of two SH-groups-the exposed and the semi-buried one-lowers enzymic activity to 70% of the initial value. Syncatalytic modication of a SH-group observed in aspartate aminotransferase from pig heart cytosol does not occur in chicken enzyme.