Regulation of Escherichia coli glutamine synthetase. Evidence for the action of some feedback modifiers at the active site of the unadenylylated enzyme.

The interaction of unadenylylated form of Escherichia coli glutamine synthetase with several substrates and effectors has been examined by magnetic resonance techniques. These studies show that two manganese ions bind per enzyme subunit. From the dramatic line broadening observed in the alanine spectra in the presence of manganese and enzyme, it is concluded that the binding of alanine occurs at a site nearer one of the two manganese sites. Electron spin resonance (ESR) titration experiments suggest apparent dissociation constants of 20 and 120 muM for manganese to these sites in the presence of 1.0 mM magnesium ion. The manganese concentration dependence of the broadening of alanine suggests an affinity of 30 muM for the manganese closest to the alanine binding site. This suggests that alanine binds closer to the more tightly bound manganese ion. Glutamate appears to displace the alanine and also appears to bind close to the strongly bound manganese ion. It is proposed that alanine and glutamine bind competitively and in the same site. The binding of alanine and ATP is shown to thermodynamically interact such that the presence of one ligand increases the affinity of the enzyme for the other ligand. The presence of ATP dramatically sharpens the alanine line width when manganese and glutamine synthetase are present. Addition of ADP or phosphate alone has little effect on the alanine line width but the addition of both ADP and phosphate shows the same dramatic sharpening as the addition of ATP alone, suggesting an induced fit conformational change in the enzyme induced by ATP or by both ADP and phosphate. A binding scheme is proposed in which all feedback inhibitors of the enzyme bind in a competitive fashion with substrates.     

Temperature and requency dependence of solvent proton relaxation rates in solutions of manganese(II) carbonic anhydrase.

Longitudinal and transverse proton relaxation rates of water in solutions of manganese(II) bovine carbonic anhydrase have been measured by pulsed nuclear magnetic resonance spectrometry as a function of temperature (2-35 degrees), frequently (5-100 MHz) and pH. The pH dependence of the longitudinal relaxation rate was fitted to a sigmoidal curve with a pK value at 7.8, while the esterase activity of the manganese(II) enzyme in the hydrolysis of p-nitrophenyl acetate revealed an inflection point at pK = 8.2. The hydration number of manganese(II) carbonic anhydrase could be derived using either the frequency dependence of T1p or the T1p/T2p ratio at only one (high) frequency. Both treatments are in agreement with a model in which one water molecule is bound to the metal at high pH. At low pH the relaxation data imply that no-H20 exists in the first coordination sphere of the manganese ion. The various parameters which are responsible for the proton relaxation mechanisms have been evaluated and are compared to other manganese(II) enzyme systems. The pH dependence of the binding constant of manganese to apocarbonic anhydrase is also reported.     

Magnetic resonance study of the three-dimensional structure of creatine kinase-substrate complexes. Implications for substrate specificity and catalytic mechanism.

The paramagnetic effects of the bound manganese ion and of a covalently attached spin label on proton nuclear spin relaxation rates have been used to calculate distances for a structural model of the MnADP and creatine complexed to creatine kinase from rabbit muscle. The nucleotide and guanidino substrates are so aligned on the enzyme that the transferable phosphoryl group on one substrate is in apposition to the acceptor moiety on the second substrate. The divalent metal ion is most probably liganded to the alpha and beta phosphates of the nucleotide substrate, both in the abortive MnADP-creatine-enzyme complex and in the active MnATP-creatine-enzyme complex. The metal ion-formate distance approximately 5 A in the Mn(II)ADP-formate-creatine-enzyme complex and less than 5 A in the Co(II)ADP-formate-creatine-enzyme complex is consistent with the suggestion that the monovalent anion is binding at the site normally occupied by the transferable phosphoryl group, thus producing a complex which mimics the transition state. Although only an upper limit of the distance from Mn(II) to the guanidino substrate could be determined in the presence of formate, it could be concluded that the disposition of the guanidino substrate changes upon addition of formate, since the relative distances of the methyl and methylene group are inverted. The effect of formate and nitrate on increasing the residence time of creatine in the MnADP-creatine-enzyme complex as determined by NMR provides evidence that the complexes observed by NMR are identical with those involved in the catalytic mechanism, since a parallel effect of formate and nitrate is observed in the kinetics of the enzymatic reaction, where the dissociation constant of creatine from the abortive quaternary complex decreases in the presence of the anions as had been determined from their inhibition of the forward reaction (Milner-White, E.J., and Watts, D.C. (1971) Biochem. J. 122, 727-740). Although the guanidino substrate is not directly liganded to the divalent metal ion, the electron paramagnetic resonance spectrum of manganese in the transition state analog complexes, i.e. nitrate-ADP-guanidino substrate-enzyme, is strongly dependent on catalytic activity of the guanidino substrate. The structural differences observed by EPR among transition state analog complexes with various guanidino substrates were not reflected in distances from Mn(II) to the guanidino substrate, which were 10% and 0.3% as active as creatine. Within the experimental error of 1 A, the distances were the same. The enzyme or the enzyme-substrate complexes may be considered to exist in a number of structurally distinct conformations in equilibrium based on the EPR spectra and on the anomalous temperature-dependence of the relaxation rates of the formate proton of the transition state analog complexes...     

Nuclear magnetic resonance investigation of human erythrocytes in the presence of manganese ions. Evidence for a thermal transition

Water proton transverse relaxation was investigated in whole blood and washed erythrocytes samples, respectively, at various temperatures and manganese concentrations. Water diffusional exchange controls proton relaxation in whole blood samples at higher Mn²⁺ concentrations (20–30 mM) or in washed erythrocyte samples at low Mn²⁺ content (1–5 mM). Mn²⁺ uptake is significant in washed normal erythrocyte samples when its concentrations is about 18 mM or higher in the medium, at temperatures below about 26°C. The thermal transition as revealed by the NMR doping method represents a switch from a water exchange process, mainly seen in the higher temperature range, to a paramagnetic ion controlled water proton relaxation in the lower temperature range.     

Design of a redox-linked active metal site: manganese bound to bacterial reaction centers at a site resembling that of photosystem II

Metals bound to proteins perform a number of crucial biological reactions, including the oxidation of water by a manganese cluster in photosystem II. Although evolutionarily related to photosystem II, bacterial reaction centers lack both a strong oxidant and a manganese cluster for mediating the multielectron and proton transfer needed for water oxidation. In this study, carboxylate residues were introduced by mutagenesis into highly oxidizing reaction centers at a site homologous to the manganese-binding site of photosystem II. In the presence of manganese, light-minus-dark difference optical spectra of reaction centers from the mutants showed a lack of the oxidized bacteriochlorophyll dimer, while the reduced primary quinone was still present, demonstrating that manganese was serving as a secondary electron donor. On the basis of these steady-state optical measurements, the mutant with the highest-affinity site had a dissociation constant of approximately 1 microM. For the highest-affinity mutant, a first-order rate with a lifetime of 12 ms was observed for the reduction of the oxidized bacteriochlorophyll dimer by the bound manganese upon exposure to light. The dependence of the amplitude of this component on manganese concentration yielded a dissociation constant of approximately 1 muM, similar to that observed in the steady-state measurements. The three-dimensional structure determined by X-ray diffraction of the mutant with the high-affinity site showed that the binding site contains a single bound manganese ion, three carboxylate groups (including two groups introduced by mutagenesis), a histidine residue, and a bound water molecule. These reaction centers illustrate the successful design of a redox active metal center in a protein complex.     

Mandelate racemase from Pseudomonas putida. Magnetic resonance and kinetic studies of the mechanism of catalysis.

The interactions of mandelate racemase with divalent metal ion, substrate, and competitive inhibitors were investigated. The enzyme was found by electron paramagnetic resonance (EPR) to bind 0.9 Mn2+ ion per subunit with a dissociation constant of 8 muM, in agreement with its kinetically determined activator constant. Also, six additional Mn2+ ions were found to bind to the enzyme, much more weakly, with a dissociation constant of 1.5 mM. Binding to the enzyme at the tight site enhances the effect of Mn2+ on the longitudinal relaxation rate (1/T1p) of water protons by a factor of 11.9 at 24.3 MHz. From the frequency dependence of 1/T1p, it was determined that there are similar to 3 water ligands on enzyme-bound Mn2+ which exchange at a rate larger than or equal to 10-7 sec-1. The correlation time for enzyme-bound Mn2+-water interaction is frequency-dependent, indicating it to be dominated by the electron spin relaxation time of Mn2+. Formation of the ternary enzyme-Mn2+-mandelate complex decreases the number of fast exchanging water ligands by similar to 1, but does not affect tau-c, suggesting the displacement or occlusion of a water ligand. The competitive inhibitors D,L-alpha-phenylglycerate and salicylate produce little or no change in the enzyme-Mn2+-H2O interaction, but ternary complexes are detected indirectly by changes in the dissociation constant of the enzyme-Mn2+ complex and by mutual competition experiments. In all cases the dissociation constants of substrates and competitive inhibitors from ternary complexes determined by magnetic resonance titrations agree with K-M and K-i values determined kinetically and therefore reflect kinetically active complexes. From the paramagnetic effects of Mn2+ on 1/T1 and 1/T2 of the 13C-enriched carbons of 1-[13C]-D,L-mandelate and 2-[13C]-D,L-mandelate, Mn2+ to carboxylate carbon and Mn2+ to carbinol carbon distances of 2.93 plus or minus 0.04 and 2.71 plus or minus 0.04 A, respectively, were calculated, indicating bidentate chelation in the binary Mn2+-mandelate complex. In the active ternary complex of enzyme, Mn2+, and D,L-mandelate, these distances increase to 5.5 plus or minus 0.2 and 7.2 plus or minus 0.2 A, respectively, indicating the presence of at least 98.9% of a second sphere complex in which Mn2+, and C1 and C2 carbon atoms are in a linear array. The water relaxation data suggest that a water ligand is immobilized between the enzyme-bound Mn2+ and the carboxylate of the bound substrate. This intervening water ligand may polarize or protonate the carboxyl group. From 1/T2p the rate of dissociation of the substrate from this ternary complex (larger than or equal to 5.6 times 10-4 sec-1) is at least 52 times greater than the maximal turnover number of the enzyme (1070 sec-1), indicating that the complex detected by nuclear magnetic resonance (NMR) is kinetically competent to participate in catalysis. Relationships among the microscopic rate constants are considered.     

Influence of Mn(II) on NMR relaxation times of biological fluids

The extent that various concentrations of the paramagnetic metal ion manganese [Mn(II)] affect nuclear magnetic resonance (NMR) relaxation times was studied in vitro. Serial dilutions of Mn(II) were prepared in distilled water, 4% human serum albumin, dog plasma, dog gallbladder bile, and dog hepatic bile. T1 and T2 of each were measured at 10.7 M Hz using magnetization recovery and spin-echo radiofrequency sequences, respectively. The results show that relaxation rates (1/T1 and 1/T2) increase in a linear manner with increasing concentration of Mn(II) in all of the solutions tested. Mn(II) dissolved in dog gallbladder and hepatic bile, dog plasma, and 4% human serum albumin reduced relaxation times to a greater extent than Mn(II) in water. T1 times were reduced to a greater extent than T2 values. Thus, in T1 weighted magnetic resonance images, the NMR signal used to produce images would be more sensitive to the presence of Mn(II) in these biological fluids than in water. Furthermore, the magnitude of this in vivo effect of Mn(II) on NMR relaxation parameters depends not only on the concentration of this paramagnetic ion, but also on the constituents comprising the biological fluids (intra- and extracellular water, bile, plasma) and the nature of the chemical molecular interactions between these constituents and Mn(II).     

Intracellular Peptidase of Bac. subtilis

A peptidase was isolated from the cells of amylase-producing Bac. subtilis by means of cell lysis with egg white lysozyme, followed by freezing and thawing, salting out, dialysis and ion-exchanger column chromatography. The enzyme required manganese ion to show the enzyme activity. Also the enzyme was stable in the presence of magnesium ion. The enzyme hydrolyzed various synthetic peptides by stepwise removal of the amino terminal amino acid of peptides and thus the peptidase was found to be aminopeptidase.     

Binding of thiocyanate and cyanide to manganese(III)-reconstituted horseradish peroxidase: a 15N nuclear magnetic resonance study

Binding of thiocyanate and cyanide ions to Mn(III) protoporphyrin-apohorseradish peroxidase complex [Mn(III)HRP] was investigated by relaxation rate measurements (at 50.68 MHz) of 15N resonance of SC15N- and C15N-. At pH = 4.0 the apparent dissociation constant (KD) for thiocyanate and cyanide binding to Mn(III)HRP was deduced to be 156 and 42 mM, respectively. The pH dependence of the 15N line width as well as apparent dissociation constant for thiocyanate and cyanide binding were quantitatively analyzed on the basis of a reaction scheme in which thiocyanate and cyanide in deprotonated form bind to the enzyme in a protonated form. The binding of thiocyanate and cyanide to Mn(III)HRP was found to be facilitated by protonation of an ionizable group on the enzyme [Mn(III)HRP] with a pKa = 4.0. From competitive binding studies it was shown that iodide, thiocyanate and cyanide bind to Mn(III)HRP at the same site; however, the binding site for resorcinol is different. The apparent dissociation constant for iodide binding deduced from competitive binding studies was found to be 117 mM, which agrees very well with the iodide binding to ferric HRP. The binding of thiocyanate and cyanide was shown to be away from the metal center and the distance of the 15N of thiocyanate and cyanide from the paramagnetic manganese ion in Mn(III)HRP was found to be 6.9 and 6.6 A, respectively. Except for cyanide binding, these observations parallel with the iodide and thiocyanate ion binding to native Fe(III)HRP. Water proton relaxivity measurements showed the presence of a coordinated water molecule to Mn(III)HRP with the distance of Mn-H2O being calculated to be 2.6 A. The slow reactivity of H2O2 towards Mn(III)HRP could be attributed to the presence of water at the sixth coordination position of the manganese ion.     

113Cd NMR of Cd2+-substituted carboxypeptidase. Support for a hexa-coordinate metal ion in the presence of inhibitors

The liquid-state 113Cd NMR data of carboxypeptidase A in the presence and absence of inhibitors obtained by Gettins (Gettins, P. (1986) J. Biol. Chem. 261, 15513-15518) are analyzed in terms of whether the inhibitors displace water from Cd2+ upon binding to the protein. This question is addressed by applying the single crystal data and the methods introduced by Honkonen and Ellis (Honkonen, R. S., and Ellis, P. D. (1984) J. Am. Chem. Soc. 106, 5488-5497). Calculations based upon these data demonstrate that displacement of water by a carboxyl group should lead to significant shielding of a 113Cd resonance by approximately 100 ppm. Since the observed 113Cd chemical shifts for carboxypeptidase A are modest and deshielding (12-17 ppm), it is argued that the chemical shifts imply that water is not displaced from the Cd2+ center upon binding of inhibitors to carboxypeptidase A. Rather, the Cd2+ ion increases its coordination number from five to six upon binding of the inhibitor.     

Reaction of peroxynitrite with Mn-superoxide dismutase. Role of the metal center in decomposition kinetics and nitration

Manganese superoxide dismutase (Mn-SOD), a critical mitochondrial antioxidant enzyme, becomes inactivated and nitrated in vitro and potentially in vivo by peroxynitrite. Since peroxynitrite readily reacts with transition metal centers, we assessed the role of the manganese ion in the reaction between peroxynitrite and Mn-SOD. Peroxynitrite reacts with human recombinant and Escherichia coli Mn-SOD with a second order rate constant of 1.0 +/- 0.2 x 10(5) and 1.4 +/- 0.2 x 10(5) m(-)1 s(-)1 at pH 7.47 and 37 degrees C, respectively. The E. coli apoenzyme, obtained by removing the manganese ion from the active site, presents a rate constant <10(4) m(-)1 s(-)1 for the reaction with peroxynitrite, whereas that of the manganese-reconstituted apoenzyme (apo/Mn) was comparable to that of the holoenzyme. Peroxynitrite-dependent nitration of 4-hydroxyphenylacetic acid was increased 21% by Mn-SOD. The apo/Mn also promoted nitration, but the apo and the zinc-substituted apoenzyme (apo/Zn) enzymes did not. The extent of tyrosine nitration in the enzyme was also affected by the presence and nature (i.e. manganese or zinc) of the metal center in the active site. For comparative purposes, we also studied the reaction of peroxynitrite with low molecular weight complexes of manganese and zinc with tetrakis-(4-benzoic acid) porphyrin (tbap). Mn(tbap) reacts with peroxynitrite with a rate constant of 6.8 +/- 0.1 x 10(4) m(-)1 s(-)1 and maximally increases nitration yields by 350%. Zn(tbap), on the other hand, affords protection against nitration. Our results indicate that the manganese ion in Mn-SOD plays an important role in the decomposition kinetics of peroxynitrite and in peroxynitrite-dependent nitration of self and remote tyrosine residues.     

Calcium ion effects on cyclic adenosine 3':5'-monophosphate bindings to the plasma membrane of Dictyostelium discoideum.

The study of cell surface cyclic adenosine 3':5'-monophosphate binding to Dictyostelium discoideum amoebae indicates that Ca2+ increases the number of binding sites without significantly affecting their affinity constant(s). The effects of the ion are observed immediately (within 4 s after addition) and appear to be readily reversible. Ca2+ effects are observed at various temperatures and pH values and are not blocked by the presence of various metabolic inhibitors. Increases, and decreases, in the apparent number of cyclic nucleotide binding sites could also be effected by concanavalin A treatments which respectively stimulate, and inhibit cell differentiation.     

Determination of the number and location of the manganese binding sites of DNA quadruplexes in solution by EPR and NMR.

The paramagnetic metal ion Mn2+ has been used to probe the electrostatic potentials of a DNA quadruplex that has two quartets with an overall fold of the chair type. A quadruplex with a basket type structure has also been examined. The binding of the paramagnetic ion manganese to these quadruplex DNAs has been investigated by solution state electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopies. The EPR results indicate that the DNA aptamer, d(GGTTGGTGTGGTTGG), binds two manganese ions and that the binding constants for each of these sites is approximately 10(5) M-1. The NMR results indicate that the binding sites of the manganese are in the narrow grooves of this quadruplex DNA. The binding sites of the DNA quadruplex formed by dimers of d(GGGGTTTTGGGG) which forms a basket structure are also in the narrow groove. These results indicate that the close approach of phosphates in the narrow minor grooves of the quadruplex structures provide strong binding sites for the manganese ions and that EPR and NMR monitoring of manganese binding can be used to distinguish between the different types of quadruplex structures.     

Reduction of CuA induces a conformational change in cytochrome c oxidase from Paracoccus denitrificans.

Cytochrome c oxidase (cytochrome aa3) from Paracoccus denitrificans contains a tightly bound manganese(II) ion, which responds to reduction of the enzyme by a change in its EPR signal (Seelig et al. (1981) Biochim. Biophys. Acta 636, 162-167). In this paper, the nature of this phenomenon is studied and the bound manganese is used as a reporter group to monitor a redox-linked conformational change in the protein. A reductive titration of the cyanide-inhibited enzyme shows that the change in the manganese EPR signal is associated with reduction of CuA. The change appears to reflect a rearrangement in the rhombic octahedral coordination environment of the central Mn2+ atom and is indicative of a redox-linked conformational transition in the enzyme. The manganese is likely to reside at the interface of subunits I and II, near the periplasmic side of the membrane. One of its ligands may be provided by the transmembrane segment X of subunit I, which has been suggested to contribute ligands to cytochrome a and CuB as well. Another manganese ligand is a water oxygen, as indicated by broadening of the manganese EPR signal in the presence of H2(17)O.     

Effects on water diffusion of inhibitors affecting various transport processes in human red blood cells.

The water permeability of human red blood cells has been monitored by nuclear magnetic resonance (NMR) following exposure to inhibitors of various transport processes across their membranes. No significant inhibition of water diffusion could be detected after the treatment of red blood cells with the anion exchange transport inhibitor dihydro-4,4'-diisothiocyano-stilbene-2,2'-disulfonate (H2DIDS) or the glucose transport inhibitors diallyl-diethyl-stilbestrol (DADES), cytochalasin B, or 30 mM iodoacetamide. It is for the first time that the effects of glucose transport inhibitors has been studied in detail by the NMR approach. A special case proved to be phloretin, an inhibitor of anion, nonelectrolyte and glucose permeability. A small but statistically significant inhibition of water permeability (around 12% at 20 degrees C) was induced by exposure to 2 mM phloretin (for 60 min at 37 degrees C); after a pretreatment of cells with 12 mM N-ethylmaleimide (NEM), for 60 min at 37 degrees C, the degree of inhibition induced by phloretin increased (becoming 17% at 20 degrees C). None of the inhibitors prevented or potentiated the strong inhibitory effect on water diffusion of a mercurial, p-chloromercuribenzene sulfonate (PCMBS). No increase in the activation energy of water diffusion occurred by treatment with the reagents used (exception the effect of PCMBS). The present results clarify some conflicting reports concerning the effects on water permeability of inhibitors of various transport processes in red blood cells and indicate that in addition to the drastic inhibition induced by mercurials other reagents may also have inhibitory effects.     

Regulation of Manganese Accumulation and Exchange in Bacillus subtilis W23

An overnight culture of Bacillus subtilis W23 in low-manganese tryptone broth is unable to sporulate and becomes hyperactive with regard to the manganese active transport system during stationary phase. When manganese is added to cells in spent or fresh medium, the cells immediately accumulate a high proportion of the manganese available in the medium. When the hyperactive cells are diluted into broth containing 10 muM Mn(2+), high intracellular manganese levels are reached, and inhibition of ribonucleic acid and protein synthesis occurs. This inhibition is relieved when the intracellular manganese concentration declines to the nontoxic levels characteristic of cells growing in 10 muM Mn(2+). The release of the accumulated manganese is achieved by a reduction in the uptake rate for manganese while the efflux rate remains essentially constant. Inhibitors of ribonucleic acid and protein synthesis prevent the reduction of the high rate of manganese uptake and, therefore, high net concentrations of manganese are maintained in the presence of these inhibitors. The hyperactive manganese uptake system is temperature dependent and inhibited by cyanide and m-chlorophenyl carbonylcyanide hydrazone.     

Biomimetic degradation of lignin and lignin model compounds by synthetic anionic and cationic water soluble manganese and iron porphyrins.

The biomimetic oxidation of 5-5' condensed and diphenylmethane lignin model compounds with several water soluble anionic and cationic iron and manganese porphyrins in the presence of hydrogen peroxide is reported. The oxidative efficiency of manganese and iron meso-tetra(2,6-dichloro-3-sulphonatophenyl) porphyrin chloride (TDCSPPMnCl and TDCSPPFeCl, respectively), meso-tetra-3-sulphonatophenyl porphyrin chloride (TSPPMnCl) and manganese meso-tetra(N-methylpyridinio)porphyrin pentaacetate (TPyMePMn(CH3COO)5) was compared on the basis of the oxidation extent of the models tested. Manganese porphyrins were found more effective in degrading lignin substructures than iron ones. Among them the cationic TPyMePMn (CH3COO)5, never used before in lignin oxidation, showed to be the best catalyst. The catalytic activity of porphyrins in hydrogen peroxide oxidation of residual kraft lignin was also investigated. The use of quantitative 31P NMR allowed the focusing on the occurrence of different degradative pathways depending on the catalyst used. TPyMePMn(CH3COO)5 was able to perform the most extensive degradation of the lignin structure, as demonstrated by the decrease of aliphatic hydroxyl groups and carboxylic acids. Noteworthy, no significant condensation reactions occurred during manganese porphyrins catalyzed oxidations of residual kraft lignin, while in the presence of iron porphyrins a substantial increase of condensed substructures was detected.     

Optical and magnetic resonance studies of formate binding to horse liver catalase and sperm whale myoglobin.

The binding of formate ion, a substrate for the peroxidatic reaction of catalase, has been investigated by magnetic resonance techniques. Comparative studies of formate binding to ferric myoglobin have also been performed. The nuclear magnetic relaxation (NMR) rate of formate and water protons is enhanced by the presence of ferric horse liver catalase. The enhancement is not changed significantly by the addition of cyanide, indicating that water and formate are still bound in the presence of cyanide. Formate proton to heme iron distances determined by magnetic resonance techniques indicate that formate does not directly bind to the heme iron of catalase or myoglobin but to the globin, and NMR relaxation occurs as a result of outersphere mechanisms. Evidence that water forms an innersphere complex with the iron atom of the catalase heme is presented. In similar experiments with ferric myoglobin, the addition of cyanide caused a large decrease in the enhancement of the proton relaxation rate of both formate and water, indicating the displacement of water and formate from the heme and the vicinity of the heme, respectively. Broad, high-spin, ferric ion electron paramagnetic resonance absorptions of catalase and myoglobin at room temperature obtained in the presence and absence of formate show that formate does not alter appreciably the heme environment of catalase or myoglobin or the spin state of the heme iron. Studies on the binding of formate to catalase as monitored by changes in the heme absorption spectrum in the visible region show one-to-one stoichiometry with heme concentration. However, the small changes observed in the visible region of the optical spectrum on addition of formate ion are attributed to a secondary effect of formate on the heme environment, rather than direct binding of formate to the heme moiety.     

Overproduction of Thermus sp. YS 8-13 manganese catalase in Escherichia coli production of soluble apoenzyme and in vitro formation of active holoenzyme.

Overproduction of Thermus sp. YS 8-13 manganese catalase in Escherichia coli BL21(DE3) was accomplished by introducing a derivative of pET-23a(+) containing a copy of the coding gene into the multicloning site. E. coli BL21(DE3)/pETMNCAT produced abundant quantities of manganese catalase as insoluble inclusion bodies. Regeneration of active catalase was achieved by denaturation in guanidine hydrochloride and subsequent dialysis in the presence of manganese ion. When the E. coli chaperone genes GroEL, GroES, DnaK, DnaJ and GrpE were coexpressed with manganese catalase, a significant fraction of the overproduced protein was partitioned into the soluble fraction. However, almost all of the soluble enzyme was isolated in a manganese-deficient apo form which could subsequently be converted into active holoenzyme by incubation with manganese ion at high temperatures. Further experiments on this apo catalase suggested that the structure of this protein was virtually identical to the active holoenzyme.     

Direct measurement of temperature-dependent solvation forces between DNA double helices.

The assembly of double stranded DNA helices with divalent manganese ion is favored by increasing temperature. Direct force measurements, obtained from the osmotic stress technique coupled with x-ray diffraction, show that the force characteristics of spontaneously precipitated Mn(2+)-DNA closely resemble those observed previously by us for other counterion condensed DNA assemblies. At temperatures below the critical one for spontaneous assembly, we have quantitated the changes in entropy and manganese ion binding associated with the transition from repulsive to attractive interactions between helices mediated by osmotic stress. The release of structured water surrounding the DNA helix to the bulk solution is the most probable source of increased entropy after assembly. Increasing the water entropy of the bulk solution by changing the manganese salt anion from CI- to ClO4- predictably and quantitatively increases the transition entropy. This is further evidence for the dominating role of water in the close interaction of polar surfaces.