Cooperativity in the dissociation of nitric oxide from hemoglobin.

The dissociation of nitric oxide from hemoglobin, from isolated subunits of hemoglobin, and from myoglobin has been studied using dithionite to remove free nitric oxide. The reduction of nitric oxide by dithionite has a rate of 1.4 X 10(3) M-1 S-1 at 20 degrees in 0.05 M phosphate, pH 7.0, which is small compared with the rate of recombination of hemoglobin with nitric oxide (25 X 10(6) M-1 S-1 (Cassoly, R., and Gibson, Q. H. (1975) J. Mol. Biol. 91, 301-313). The rate of NO combination with chains and myoglobin was found to be 24 X 10(6) M-1 S-1 and 17 X 10(6) M-1 S-1, respectively. Hence, the observed progress curve of the dissociation of nitric oxide is dependent upon the dithionite concentration and the total heme concentration. Addition of excess carbon monoxide to the dissociation mixture reduces the free heme yielding a single exponential process for chains and for myoglobin which is dithionite and heme concentration independent over a wide range of concentrations. The rates of dissociation of nitric oxide from alpha chains, from beta chains, and from myoglobin are 4.6 X 10(-5) S-1, 2.2 X 10(-5) S-1, and 1.2 X 10(4) S-1, respectively, both in the presence and in the absence of carbon monoxide at 20 degrees in 0.05 M phosphate, pH 7.0. Analogous heme and dithionite concentration dependence is found for the dissociation of nitric oxide from tetrameric hemoglobin. The reaction is cooperative, the intrinsic rate constants for the dissociation of the 1st and 4th molecules of NO differing about 100-fold. With hemoglobin, replacement of NO by CO at neutral pH is biphasic in phosphate buffers. The rate of the slow phase is 1 X 10(-5) S-1 and is independent of pH. The amplitude of the fast phase increases with lowering of pH. By analogy with the treatment of the HbCO + NO reaction given by Salhany et al. (Salhany, J.M., Ogawa, S., and Shulman, R.G. (1975) Biochemistry 14, 2180-2190), the fast phase is attributed to the dissociation of NO from T state molecules and the slow phase to dissociation from R state molecules. Analysis of the data gives a pH-independent value of 0.01 for the allosteric constant c (c = Kr/Kt where Kr and Kt are the dissociation constants for NO from the R and T states, respectively) and pH-dependent values of L (2.5 X 10(7) at pH 7 in 0.05 M phosphate buffer). The value of c is considerably greater than that for O2 and CO. Studies of the difference spectrum induced in the Soret region by inositol hexaphosphate are also reported. This spectrum does not arise directly from the change of conformation between R and T states. The results show that if the equilibrium binding curve for NO could be determined experimentally, it would show cooperativity with Hill's n at 50% saturation of about 1.6.     

Studies of human hemoglobin intermediates. The double mixing method for studying the reactions of the species Hb4(CO) and Hb4(CO)2

Using the double mixing method we have studied the reactions of the partially liganded species (Hb4, Hb4L1, Hb4L2, Hb4L3) of normal human hemoglobin with carbon monoxide. In the first mixing, oxygen is removed from the species Hb4(O2) chi (CO) gamma and at the second mixing the species Hb4(CO) gamma reacts with CO. At 90% saturation of oxyHb with CO the main intermediate species are Hb4(CO)3 and Hb4(CO)2, and at 10% saturation Hb4 and Hb4(CO). The four CO-combination rate constants determined are: l'1 = 1 X 10(5) M-1 S-1, l'2 = 7 X 10(5) M-1 S-1, l'3 = 2 X 10(5) M-1 S-1 and l'4 = 4.8 X 10(6) M-1 S-1. The results indicate that there is no monotonic increase in the successive CO-combination rate constants. It is difficult to explain these results on the basis of the two-state model (Monod et al., 1965) or the stereochemical model of Perutz (1970).     

Classification and localization of hemoglobin binding sites on the red blood cell membrane.

The binding of hemoglobin to the red cell membrane was characterized over a wide range of free hemoglobin concentrations by measurement of membrane bound and supernatant hemoglobin. Scatchard analysis of the binding data revealed two classes of sites: high affinity sites with a binding constant of 1 X 10(8) M-1 and 1.2 X 10(6) sites per cell, and a second, low affinity class of sites with a binding constant of 6 X 10(6)M-1 and 6 X 10(6) sites per cell. The low affinity sites are shown to be nonspecific and appear to be a result of the ghost preparation. The high affinity sites are shown to be specific to the inner surface of the red cell membrane. The competition of hemoglobin and glyceraldehyde-3-phosphate dehydrogenase suggests band III proteins as a potential binding site for hemoglobin.     

Action mechanism of Escherichia coli DNA photolyase. I. Formation of the enzyme-substrate complex

Escherichia coli DNA photolyase (photoreactivating enzyme) is a flavoprotein. The enzyme binds to DNA containing pyrimidine dimers in a light-independent step and, upon illumination with 300-600 nm radiation, catalyzes the photosensitized cleavage of the cyclobutane ring thus restoring the integrity of the DNA. We have studied the binding reaction using the techniques of nitrocellulose filter binding and flash photolysis. The enzyme binds to dimer-containing DNA with an association rate constant k1 estimated by two different methods to be 1.4 X 10(6) to 4.2 X 10(6) M-1 S-1. The dissociation of the enzyme from dimer-containing DNA displays biphasic kinetics; for the rapidly dissociating class of complexes k2 = 2-3 X 10(-2) S-1, while for the more slowly dissociating class k2 = 1.3 X 10(-3) to 6 X 10(-4) S-1. The equilibrium association constant KA, as determined by the nitrocellulose filter binding assay and the flash photolysis assay, was 4.7 X 10(7) to 6 X 10(7) M-1, in reasonable agreement with the values predicted from k1 and k2. From the dependence of the association constant on ionic strength we conclude that the enzyme contacts no more than two phosphodiester bonds upon binding; this strongly suggests that the pyrimidine dimer is the main structural determinant of specific photolyase-DNA interaction and that nonspecific ionic interactions do not contribute significantly to substrate binding.     

Functional and subunit assembly properties of hemoglobin Alberta (alpha 2 beta 2(101) Glu----Gly)

Hemoglobin Alberta has an amino acid substitution at position 101 (Glu----Gly), a residue involved in the alpha 1 beta 2 contact region of both the deoxy and oxy conformers of normal adult hemoglobin. Oxygen equilibrium measurements of stripped hemoglobin Alberta at 20 degrees C in the absence of phosphate revealed a high affinity (P50 = 0.75 mm Hg at pH 7), co-operative hemoglobin variant (n = 2.3 at pH 7) with a normal Bohr effect (- delta log P50/delta pH(7-8) = 0.65). The addition of inositol hexaphosphate resulted in a decrease in oxygen affinity (P50 = 8.2 mm Hg at pH 7), a slight increase in the value of n and an enhanced Bohr effect. Rapid mixing experiments reflected the equilibrium results. A rapid rate of carbon monoxide binding (l' = 7.0 X 10(5) M-1 S-1) and a slow rate of overall oxygen dissociation (k = 15 s-1) was seen at pH7 and 20 degrees C in the absence of phosphate. Under these experimental conditions the tetramer stability of liganded and unliganded hemoglobin Alberta was investigated by spectrophotometric kinetic techniques. The 4K4 value (the liganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta was found to be 0.83 X 10(-6) M compared to a 4K4 value for hemoglobin A of 2.3 X 10(-6) M, indicating that the Alberta tetramer was less dissociated into dimers than the tetramer of hemoglobin A. The values of 0K4 (the unliganded tetramer-dimer equilibrium dissociation constant) for hemoglobin Alberta and hemoglobin A were also measured and found to be 2.5 X 10(-8) M and 1.5 X 10(-10) M, respectively, demonstrating a greatly destabilized deoxyhemoglobin tetramer for hemoglobin Alberta compared to deoxyhemoglobin A. The functional and subunit dissociation properties of hemoglobin Alberta appear to be directly related to the dual role of the beta 101 residue in stabilizing the tetrameric form of the liganded structure, while concurrently destabilizing the unliganded tetramer molecule.     

Kinetics of carbon monoxide binding to fully and partially reduced human hemoglobin valency hybrids.

The kinetics of carbon monoxide binding following fast reduction of the valency hybrids alpha2+betaCO2 and alphaCO2beta+2 by hydrated electrons have been studied at different degrees of reduction. The results show that at pH 6.0 and 7.0 reduction of one heme group yields a species which reacts fast with carbon monoxide (rate constant of the order of 10(6) M-1S-1). At pH 6.0 the intermediates alphaCO2beta2 and alpha2betaCO2 bind carbon monoxide with a rate characteristic of the T state. At pH 7.0 alphaCO2beta2 is for the greater part in the T state, while in the case of alpha2betaCO2 the R and the T state are about equally populated.     

Kinetic and equilibrium studies of the reactions of heme-substituted horse heart myoglobins with oxygen and carbon monoxide.

In order to study the effects of chemical modifications of the vinyl groups of heme on oxygen and carbon monoxide binding to myoglobin, apomyoglobins from horse heart were reconstituted with six different hemins with various side chains. Laser flash photolysis experiments of these reconstituted myoglobins showed that the combination rate constants for oxygen (k') and carbon monoxide (l') were closely related to the electron-attractive properties of the side chains. The k' values obtained in 0.1 M potassium phosphate buffer, pH 7.0, at 20 degrees were 0.83 (meso-), 2.4 (deutero-), 1.1 (reconstituted proto-), 1.2 (native proto-), 1.5 (2-formyl-4-vinyl-), 1.9 (2-vinyl-4-formyl-), and 2.7 X 10(7) M-1 S-1 (2,4-diformylmyoglobins), and the corresponding l' values were 2.8, 18, 4.8, 5.1, 7.1, 15, and 35 X 10(5) M-1 S-1, respectively. These rate constants tend to increase as the electron-withdrawing power of the side chains increases, indicating that reduced electron density of the iron atom of heme in myoglobin favors the combination reaction for both oxygen and carbon monoxide. Equilibrium constants (L) between carbon monoxide and various myoglobins were also determined by measuring the partition coefficients (M) between oxygen and carbon monoxide for the myoglobins, and were also found to be closely related to the electronic properties (pK3 of porphyrin) of the heme side chains. The equilibrium association constants for carbon monoxide thus obtained increased with a decrease in pK3 value of the porphyrin. This order was completely opposite to the case of the oxygen binding reaction. The dissociation rate constants for oxygen (k) and carbon monoxide (l) were calculated from the equilibrium and the combination rate constants. The dissociation rate constants showed a similar characteristic to the combination rate constants and increased with the increase in electron attractivity of heme side chains. The concomitant increase in both the combination and dissociation rate constants with increase in electronegativity of the iron atom suggests that these reactions have different rate determining steps, although such a reaction process is contradictory to the generally accepted concept that in a reversible reaction, both on and off reactions proceed through the same transition state. In the on reaction sigma bond formation appears to be dominant, while in the off reaction eta bond break-up is more important.     

Kinetics of oxygen and carbon monoxide binding to liver fluke (Dicrocoelium dendriticum) hemoglobin. An extreme case?

The kinetics of oxygen and carbon monoxide binding to the monomeric liver fluke (Dicrocoelium dendriticum) hemoglobin have been studied. The ligand association rates are approximately 1 X 10(8) and approximately 3 X 10(8) M-1 s-1, respectively, for CO and O2 and show no pH dependence. On the contrary the ligand dissociation rates decrease by lowering the pH below 7, the pK of the transition being around 5.5. These findings, together with spectroscopic properties of the protein, are discussed in relation to the fact that, in this hemoglobin, the distal histidine is replaced by a glycine.     

Properties of passive binding of calcium to endoplasmic reticulum from adipocytes.

Calcium binding to isolated adipocyte microsomes enriched in endoplasmic reticulum has been characterized. Binding was concentration-dependent, saturable, and totally dissociable. Steady state was reached within 20 min at all calcium concentrations tested. Three apparent classes of binding sites were identified in kinetic and steady state studies using calcium concentrations from 1 muM to 10 mM. The affinity constants (and maximum binding capacities) as determined by computer analysis for the three classes were 2.1 X 10(5) M-1 (0.28 nmol of calcium/mg of protein), 1.3 X 10(4) M-1 (1.1 nmol/mg), and 1.3 X 10(2) M-1 (35 nmol/mg). The dissociation rate constants for the high and intermediate affinity classes of sites were 1.6 X 10(-3) S-1, respectively, and the association rate constant for the high affinity sites was 8 X 10(2) M-1 S-1. The affinity constant calculated from the rate constants was 5.0 X 10(5) M-1 for the high affinity sites in agreement with the value obtained in studies at steady state. The three classes of binding sites were specific for calcium. Magnesium was a noncompetitive inhibitor of calcium binding to all three classes of sites with a Ki of 9 to 12 mM. Calcium binding at 1 muM calcium was 50% inhibited by 18 muM La3+, 600 muM Sr2+, or 2.7 mM Ba2+. These data represent the first analysis of passive calcium binding to endoplasmic reticulum from nonmuscular cells and the first report of corresponding rate constants for either endoplasmic or sarcoplasmic reticulum. The characteristics of the binding are consistent with the properties of calcium transport by endoplasmic reticulum of adipocytes. The characteristics and specificity of the calcium binding constitute further evidence that endoplasmic reticulum plays an important role in cellular calcium homeostasis.     

Fluorescence stopped flow analysis of the interaction of virginiamycin components and erythromycin with bacterial ribosomes

The kinetics of the interaction between the 50 S subunits (R) of bacterial ribosomes and the antibiotics virginiamycin S (VS), virginiamycin M (VM), and erythromycin have been studied by stopped flow fluorimetric analysis, based on the enhancement of VS fluorescence upon its binding to the 50 S ribosomal subunit. Virginiamycin components M and S exhibit a synergistic effect in vivo, which is characterized in vitro by a 5- to 10-fold increase of the affinity of ribosomes for VS, and by the loss of the ability of erythromycin to displace VS subsequent to the conformational change (from R to R*) produced by transient contact of ribosomes with VM. Our kinetic studies show that the VM-induced increase of the ribosomal affinity for VS (K*VS = 25 X 10(6) M-1 instead of KVS = 5.5 X 10(6) M-1) is due to a decrease of the dissociation rate constant (k*-VS = 0.008 s-1 instead of 0.04 s-1). The association rate constant remains practically the same (k+VS approximately k*+VS = 2.8 X 10(5) M-1 s-1), irrespective of the presence of VM. VS and erythromycin bind competitively to ribosomes. This effect has been exploited to determine the dissociation rate constant of VS directly by displacement experiments from VS . 50 S complexes, and the association rate constant of erythromycin (k+Ery = 3.2 X 10(5) M-1 S-1) on the basis of competition experiments for binding of free erythromycin and VS to ribosomes. By making use of the change in competition behavior of erythromycin and VS, after interaction of ribosomes with VM, the conformational change induced by VM has been explored. Within the experimentally available concentration region, the catalytic effect of VM has been shown to be coupled to its binding kinetics, and the association rate constant of VM has been determined (k+VM = 1.4 X 10(4) M-1 S-1). Evidence is presented for a low affinity binding of erythromycin (K*Ery approximately 3.3 X 10(4) M-1) to ribosomes altered by contact with VM. A model involving a sequence of 5 reactions has been proposed to explain the replacement of ribosome-bound erythromycin by VS upon contact of 50 S subunits with VM.     

Involvement of heparin chain length in the heparin-catalyzed inhibition of thrombin by antithrombin III

The mechanism of the heparin-promoted reaction of thrombin with antithrombin III was investigated by using covalent complexes of antithrombin III with either high-affinity heparin (Mr = 15,000) or heparin fragments having an average of 16 and 12 monosaccharide units (Mr = 4,300 and 3,200). The complexes inhibit thrombin in the manner of active site-directed, irreversible inhibitors: (Formula: see text) That is, the inhibition rate of the enzyme is saturable with respect to concentration of complexes. The values determined for Ki = (k-1 + k2)/k1 are 7 nM, 100 nM, and 6 microM when the Mr of the heparin moieties are 15,000, 4,300, 3,200, respectively, whereas k2 (2 S-1) is independent of the heparin chain length. The bimolecular rate constant k2/Ki for intact heparin is 3 X 10(8) M-1 S-1 and the corresponding second order rate constant k1 is 6.7 X 10(8) M-1 S-1, a value greater than that expected for a diffusion-controlled bimolecular reaction. The bimolecular rate constants for the complexes with heparin of Mr = 4,300 and 3,200 are, respectively, 2 X 10(7) M-1 S-1 and 3 X 10(5) M-1 S-1. Active site-blocked thrombin is an antagonist of covalent antithrombin III-heparin complexes: the effect is monophasic and half-maximum at 4 nM of antagonist against the complex with intact heparin, whereas the effect is weaker against complexes with heparin fragments and not monophasic. We conclude that virtually all of the activity of high affinity, high molecular weight heparin depends on binding both thrombin and antithrombin III to heparin, and that the exceptionally high activity of heparin results in part from the capacity of thrombin bound nonspecifically to heparin to diffuse in the dimension of the heparin chain towards bound antithrombin III. Increasing the chain length of heparin results in an increased reaction rate because of a higher probability of interaction between thrombin and heparin in solution.     

Oxygen binding to dithionite-reduced chloroperoxidase

Both the kinetics of ferric chloroperoxidase reduction by dithionite and the binding of molecular oxygen to ferrous chloroperoxidase have been studied. The oxyferrous chloroperoxidase decays spontaneously to the ferric enzyme. In addition the corresponding rapid-scan spectra have been recorded. The reduction reaction is caused by SO-.2 with a rate constant of (7.7 +/- 1.0) X 10(4) M-1 S-1. Oxygen binding occurs with a rate constant of (5.5 +/- 1.0) X 10(5) M-1 S-1 over the pH range 3.5-6. Oxyferrous chloroperoxidase has a Soret absorption peak at 428 nm and two partially resolved peaks at 555 nm and 588 nm. Isosbestic points occur at the following wavelengths: between ferrous and oxyferrous chloroperoxidase at 419, 545, 555 and 580 nm; between oxyferrous and ferric chloroperoxidase at 419, 487, 540, 609 and 682 nm.     

Kinetic properties of binding of myosin subfragment-1 with F-actin in the absence of nucleotide

The rate constant for the binding of myosin subfragment-1 (S-1) with F-actin in the absence of nucleotide, k1, and that for dissociation of the F-actin-myosin subfragment-1 complex (acto-S-1), k-1, were measured independently. The rate of S-1 binding with F-actin was measured from the time course of the change in the light scattering intensity after mixing S-1 with various concentrations of F-actin and k1 was found to be 2.55 X 10(6) M-1 X S-1 at 20 degrees C. The dissociation rate of acto-S-1 was determined using F-actin labeled with pyrenyl iodoacetamide (Pyr-FA). Pyr-FA, with its fluorescence decreased by binding with S-1, was mixed with acto-S-1 complex and the rate of displacement of F-actin by Pyr-FA was measured from the decrease in the Pyr-FA fluorescence intensity. The k-1 value was calculated to be 8.5 X 10(-3) S-1 (or 0.51 min-1). The value of the dissociation constant of S-1 from acto-S-1 complex, Kd, was calculated from Kd = k-1/k1 to be 3.3 X 10(-9) M at 20 degrees C. Kd was also measured at various temperatures (0-30 degrees C), and the thermodynamic parameters, delta G degree, delta H degree, and delta S degree, were estimated from the temperature dependence of Kd to be -11.3 kcal/mol, +2.5 kcal/mol, and +47 cal/deg . mol, respectively. Thus, the binding of the myosin head with F-actin was shown to be endothermic and entropy-driven.     

Properties of NAD binding by synaptic membranes of rat brain

The binding of [14C]NAD to rat brain synaptic membranes is reversible and depends on incubation time, temperature and protein concentration in the reaction mixture. The value of the rate constant for [14C]NAD binding to the synaptic membranes at 24 degrees C (kl) is 1.1 X 10(-6) M-1 S-1, the rate constant for dissociation of the [14C]NAD-receptor complex (k-1) is 3.3 X 10(-3) S-1. The value of the constant for the ligand dissociation from this complex (Kd) is 3.0 nmole. Treatment of the experimental results in the Scatchard plots for the equilibrium binding of [14C]NAD to the synaptic membranes demonstrated that the receptor sites with high and low affinities for the ligand (Kd1 = 3.3 nmol, Kd2 = 14.4 nmole) and with binding capacities of 44 and 77 pmole of [14C]NAD, respectively. It was found that the synaptosomal membrane components which bind the labelled NAD have a protein nature. Data from [14C]NAD and [nicotinamide-3H]NAD binding suggest that brain synaptic membranes bind NAD at the nicotinamide and adenylic moieties.     

The effect of divalent metal cations on the inhibition of human coagulation factor Xa by plasma proteinase inhibitors

The inactivation of human coagulation factor Xa by the plasma proteinase inhibitors alpha 1-antitrypsin, antithrombin III and alpha 2-macroglobulin in purified systems was found to be accelerated by the divalent cations Ca2+, Mn2+ and Mg2+. The rate constant for the inhibition of factor Xa by antithrombin III rose from 2.62 X 10(4) M-1 X min-1 in the absence of divalent cations to a maximum of 6.40 X 10(4) M-1 X min-1 at 5 mM Ca2+, 8.10 X 10(4) M-1 X min-1 at 5 mM Mn2+, with a slight decrease in rate at higher cation concentrations. Mg2+ caused a gradual rise in rate constant to 5.65 X 10(4) M-1 X min-1 at 20 mM. The rate constant for the inhibition of factor Xa by alpha 1-antitrypsin in the absence of divalent cations was 5.80 X 10(3) M-1 X min-1. Ca2+ increased the rate to 1.50 X 10(4) M-1 X min-1 at 5 mM and Mn2+ to 2.40 X 10(4) M-1 X min-1 at 6 mM. The rate constant for these cations again decreased at higher concentrations. Mg2+ caused a gradual rise in rate constant to 1.08 X 10(4) M-1 X min-1 at 10 mM. The rate constant for the factor Xa-alpha 2-macroglobulin reaction was raised from 6.70 X 10(3) M-1 X min-1 in the absence of divalent cations to a maximum of 4.15 X 10(4) M-1 X min-1 at 4 mM Ca2+, with a decrease to 3.05 X 10(4) M-1 at 10 mM. These increases in reaction rate were correlated to the binding of divalent cations to factor Xa by studying changes in the intrinsic fluorescence and dimerization of factor Xa. The changes in fluorescence suggested a conformational change in factor Xa which may be responsible for the increased rate of reaction, whilst the decrease in rate constant at higher concentrations of Ca2+ and Mn2+ may be due to factor Xa dimerization.     

Elementary steps in the reaction of the pyruvate dehydrogenase complex from pig heart. Kinetics of thiamine diphosphate binding to the complex

In the progress curve of the reaction of the pyruvate dehydrogenase complex, a lag phase was observed when the concentration of thiamin diphosphate was lower than usual (about 0.2-1 mM) in the enzyme assay. The length of the lag phase was dependent on thiamin diphosphate concentration, ranging from 0.2 min to 2 min as the thiamin diphosphate concentration varied from 800 nM to 22 nM. The lag phase was also observed in the elementary steps catalyzed by the pyruvate dehydrogenase component. A Km value of 107 nM was found for thiamin diphosphate with respect to the steady-state reaction rate following the lag phase. The pre-steady-state kinetic data indicate that the resulting lag phase was the consequence of a slow holoenzyme formation from apoenzyme and thiamin diphosphate. The thiamin diphosphate can bind to the pyruvate dehydrogenase complex in the absence of pyruvate, but the presence of 2 mM pyruvate increases the rate constant of binding from 1.4 X 10(4) M-1 S-1 to 1.3 X 10(5) M-1 S-1 and decreases the rate constant of dissociation from 2.3 X 10(-2) S-1 to 4.1 X 10(-3) S-1. On the other hand, the effect of pyruvate on the thiamin diphosphate binding revealed the existence of a thiamin-diphosphate-independent pyruvate-binding site in the pyruvate dehydrogenase complex. Direct evidence was also obtained with fluorescence techniques for the existence of this binding site and the dissociation constant of pyruvate was found to be 0.38 mM. On the basis of these data we have proposed a random mechanism for the binding of pyruvate and thiamin diphosphate to the complex. Binding of substrates to the enzyme complex caused an increase in the fluorescence of the dansylaziridine-labelled pyruvate dehydrogenase complex, showing that binding of substrates to the complex is accompanied by structural changes.     

Kinetics of reduction of Clostridium pasteurianum rubredoxin by laser photoreduced spinach ferredoxin:NADP+ reductase and free flavins. Electron transfer within a protein-protein complex

The kinetics of reduction of oxidized Clostridium pasteurianum rubredoxin (Rdox) by free flavin semiquinones generated by the laser flash photolysis technique and by spinach ferredoxin:NADP+ reductase (FNR) semiquinone (also produced by flavin semiquinone reduction) have been investigated under anaerobic conditions. 5-Deazariboflavin semiquinone (5-dRf) rapidly reduces oxidized rubredoxin (Rdox) (k = 3.0 X 10(8) M-1 S-1) and oxidized ferredoxin:NADP+ reductase (FNRox) to the semiquinone level (k = 5.5 X 10(8) M-1 S-1). Lumiflavin semiquinone reduces Rdox more slowly (k = 1.3 X 10(7) M-1 S-1) and is not measurably reactive with FNRox. Absorption difference spectroscopy and difference CD indicate that Rdox and FNRox form a 1:1 complex at low ionic strength (10 mM), which is completely dissociated at higher ionic strength (310 mM). Apparent second order rate constants for reduction of Rdox in its free and complexed state by lumiflavin semiquinone are the same. Reduction of Rdox (both free and complexed) by free FNR semiquinone and intracomplex electron transfer were investigated using 5-dRf as the reductant. At I = 10 mM, a first order rate constant of 2.0 X 10(3) S-1 was obtained, which corresponds to the processes involved in intracomplex electron transfer from FNR semiquinone to Rdox. A second order reaction between free FNR semiquinone and complexed Rdox was also observed to occur (k = 5 X 10(7) M-1 S-1). At I = 310 mM, these reactions are not observed and the reaction of FNR semiquinone with free Rdox is second order (k = 4 X 10(6) M-1 S-1).     

Kinetics of cyanide binding to Chromatium vinosum ferricytochrome c'

The kinetics of the reversible binding of cyanide by the ferric cytochrome c' from Chromatium vinosum have been studied over the pH range 6.9-9.6. The reaction is extremely slow at neutral pH compared to the reactions of other high-spin ferric heme proteins with cyanide. The observed bimolecular rate constant at pH 7.0 is 2.25 X 10(-3) M-1 s-1, which is approximately 10(7)-fold slower than that for peroxidases, approximately 10(5)-fold slower than those for hemoglobin and myoglobin, and approximately 10(2)-fold to approximately 10(3)-fold slower than that recently reported for the Glycera dibranchiata hemoglobin, which has anomalously slow cyanide rate constants of 4.91 X 10(-1), 3.02 X 10(-1), and 1.82 M-1 s-1 for components II, III, and IV, respectively [Mintorovitch, J., & Satterlee, J. D. (1988) Biochemistry 27, 8045-8050; Mintorovitch, J., Van Pelt, D., & Satterlee, J. D. (1989) Biochemistry 28, 6099-6104]. The unusual ligand binding property of this cytochrome c' is proposed to be associated with a severely hindered heme coordination site. Cyanide binding is also characterized by a nonlinear cyanide concentration dependence of the observed rate constant at higher pH values, which is interpreted as involving a change in the rate-determining step associated with the formation of an intermediate complex between the cytochrome c' and cyanide prior to coordination. The pH dependence of both the binding constant for the formation of the intermediate complex and the association rate constant for the subsequent coordination to the heme can be attributed to the ionization of HCN, where cyanide ion binding is the predominant process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Active-site-directed inactivators of the Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase of Streptomyces albus G

Several types of active-site-directed inactivators (inhibitors) of the Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase were tested. (i) Among the heavy-atom-containing compounds examined, K2Pt(C2O4)2 inactivates the enzyme with a second-order rate constant of about 6 X 10(-2)M-1 X S-1 and has only one binding site located close to the Zn2+ cofactor within the enzyme active site. (ii) Several compounds possessing both a C-terminal carboxylate function and, at the other end of the molecule, a thiol, hydroxamate or carboxylate function were also examined. 3-Mercaptopropionate (racemic) and 3-mercaptoisobutyrate (L-isomer) inhibit the enzyme competitively with a Ki value of 5 X 10 X 10(-9)M. (iii) Classical beta-lactam compounds have a very weak inhibitory potency. Depending on the structure of the compounds, enzyme inhibition may be competitive (and binding occurs to the active site) or non-competitive (and binding causes disruption of the protein crystal lattice). (iv) 6-beta-Iodopenicillanate inactivates the enzyme in a complex way. At high beta-lactam concentrations, the pseudo-first-order rate constant of enzyme inactivation has a limit value of 7 X 10(-4)S-1 X 6-beta-Iodopenicillanate binds to the active site just in front of the Zn2+ cofactor and superimposes histidine-190, suggesting that permanent enzyme inactivation is by reaction with this latter residue.     

Occurrence and properties of a prostaglandin F2alpha receptor in bovine corpora lutea.

Prostaglandin F2alpha was specifically bound by a particulate fraction from bovine corpora lutea. The rate constants for the association (7.5 X 10(3) M-1 S-1) and dissociation (2.1 X 10-4 S-1) reactions gave a dissociation constant of 2.8 X 10(-8) M which is similar to that determined from a Scatchard plot of binding data at equilibrium (5 X 10(-8) M). The receptor was stable for several hours at 23 degrees C but was rapidly destroyed at 37 degrees C. The pH optimum for the binding reaction was 6.3. The receptor had high specificity for prostaglandin F2alpha and had much lower affinities for other prostaglandins. Luteinizing and follicle-stimulating hormones had no effect on the prostaglandin F2alpha-receptor interaction.