The importance of monopole-monopole and monopole-dipole interactions on the binding of NADPH and NADPH analogues to p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. Effects of pH and ionic strength.

NADPH binding to p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens is found to be strongly dependent on pH and ionic strength. In the ionic strength range of 0.02-0.15 M, optimal NADPH binding is observed at a pH value of 6.4. Extrapolation of the dissociation constants to infinite ionic strength shows that under these conditions optimal binding occurs at pH values greater than 8. Similar results were obtained for complexes between the enzyme and two NADPH analogues in the presence or absence of the substrate. The experimental data can be explained by a theoretical model in which monopole-monopole or monopole-dipole interactions between the enzyme and the ligand are dominant. Changes in the former interaction prevail at low ionic strength and low pH values while the changes in the latter prevail at high ionic strength and high pH values. The dipole moment of the enzyme in the direction of the NADPH binding site was calculated from the ionic strength and pH dependence of the complex formation. The calculated dipole moment of the enzyme is about 2000 Debye at pH 6 and decreases to about 1100 Debye at pH 8.5. The results are discussed with respect to published results, including data obtained from the enzyme from a different source.

A pulse-radiolysis study of the reduction of flavodoxin from Megasphaera elsdenii by viologen radicals. A conformational change as a possible regulating mechanism.

Pulse-radiolysis experiments were performed on solutions containing methyl or benzyl viologen and flavodoxin. Viologen radicals are formed after the pulse. The kinetics of the reaction of these radicals with flavodoxin were studied. The kinetics observed depend strongly on the concentration of oxidized viologen. Therefore one must conclude that a relatively stable intermediate is formed after the reduction of flavodoxin. The midpoint potential of the intermediate state is -(480 +/- 30) mV, and is hardly dependent on the pH between 7 and 9.2. Due to a conformational change (k2 approximately equal to 10(5)S-1) the intermediate state decays to the stable semiquinone form of flavodoxin. The delta G of the conformational change at pH 8 is about 29 kJ mol -1 (0.3 eV). This means that the upper limit for the pK of N-5 in the semiquinone form will be 13. The activation energy of the conformational change is 43 kJ mol -1 (0.45 eV). The reaction between methyl viologen radicals and the semiquinone of flavodoxin can be described by a normal bimolecular reaction. The reaction is diffusion-controlled with a forward rate constant of (7 +/- 1) X 10(8) M -1S -1 (pH 8, I = 55 mM). The midpoint potential of the semiquinone/hydroquinone was found to be -(408 +/- 5) mV. A consequence of the intermediate state is that flavodoxin (Fld) could be reduced by a two-electron process, the midpoint potential of which should be located between -440 mV less than Em (Fld/FldH-) less than -290 mV. The exact value will depend on the delta G of the conformational change between the fully reduced flavodoxin with its structure in the oxidized form and the fully reduced flavodoxin with its structure in the hydroquinone form. The conditions are discussed under which flavodoxin could behave as a two-electron donor.

An estimate of the dipole moment of superoxide dismutase.

A lower limit for the value of the dipole moment of superoxide dismutase (SOD) is calculated to be 485 Debye. This limit follows from the observation that the rate constant of the reaction between superoxide (O-2) and SOD decreases upon increasing the ionic strength, and the fact that at pH greater than 5 SOD has a net negative charge.

A pulse-radiolysis study of cytochrome c3. Kinetics of the reduction of cytochrome c3 by methyl viologen radicals and the characterisation of the redox properties of cytochrome c3 from Desulfovibrio vulgaris (Hildenborough).

1. Pulse-radiolysis experiments were performed in the presence of methyl viologen and cytochrome c3. After the pulse, methyl viologen radicals are formed and the kinetics of these radicals with cytochrome c3 are studied, The reaction between cytochrome c3 and methyl viologen radicals (MV+) is diffusion controlled. The ionic strength dependence and the pH-dependence of this reaction were studied. From the ionic strength dependence (at pH 7.8) we found that the net charge of the fully oxidized cytochrome c3 molecule was Z = + 4.7 +/- 0.7. 2. After the pulse an equilibrium is reached for the reaction of MV+ with cytochrome c3. From this equilibrium an apparent midpoint potential can be obtained. The apparent midpoint potential of this multihaem molecule was found to depend on the degree of reduction, alpha. With the help of the Nernst equation an empirical equation is obtained to describe this dependence of the midpoint potential: E0 = - 0.250 - 0.088 alpha (in V). 3. An estimation is made of the energy of interaction between the haems due to electrostatic interactions (delta epsilon less than 32 mV) and due to ionic strength effects (- 12 mV less than delta epsilon less than 26 mV). The results suggest that the redox properties of the individual haems in the cytochrome c3 molecule are dependent on the degree of reduction of the other haems in the molecule. 4. The reaction of cytochrome c3 with MV+ or with ethanol radicals (EtOH) has been compared with the reactions of horse-heart cytochrome c and of metmyoglobin with the same radicals. The reaction of MV+ or EtOH with horse-heart cytochrome c is found to be diffusion controlled; the reactions with metmyoglobin on the other hand are most probably controlled by an activation energy.

The reaction of cytochrome aa3 with (porphyrin) cytochrome c as studied by pulse radiolysis.

(1) Using the pulse-radiolysis and stopped-flow techniques, the reactions of iron-free (porphyrin) cytochrome c and native cytochrome c with cytochrome aa3 were investigated. The porphyrin cytochrome c anion radical (generated by reduction of porphyrin cytochrome c by the hydrated electron) can transfer its electron to cytochrome aa3. The bimolecular rate constant for this reaction is 2 x 10(7) M-1 . s-1 (5 mM potassium phosphate, 0.5% Tween 20, pH 7.0, 20 degrees C). (2) The ionic strength dependence of the cytochrome c-cytochrome aa3 interaction was measured in the ionic strength range between 40 and 120 mM. At ionic strengths below 30 mM, a cytochrome c-cytochrome aa3 complex is formed in which cytochrome c is no longer reducible by the hydrated electron. A method is described by which the contributions of electrostatic forces to the reaction rate can be determined. (3) Using the stopped-flow technique, the effect of the dielectric constant (epsilon) of the reaction medium on the reaction of cytochrome C with cytochrome aa3 was investigated. With increasing epsilon the second-order rate constant decreased.

The ionic strength dependence of the rate of a reaction between a small ion and a large ion with a dipole moment.

The ionic strength dependence of the rate constant of a reaction between a small ion and a large ion with a dipole moment (e.g. a protein) is described. This description takes into account only the electrostatic interactions between the two ions. This approach agrees with the Marcus theory treatment of the electrostatic interactions and also with the Debye-Hückel theory which is based on changes in the activity coefficients of the reactants. The contribution of the dipole moment of the protein to the ionic strength dependence of the rate constant has been calculated. A method is described whereby one can calculated the charge of the protein without knowing the precise ionic strength dependence of the rate constant. Two applications are mentioned to illustrate the usefulness of the method.

A non-equilibrium state of deoxyhaemoglobin. Temperature-dependence and oxygen binding.

After reduction of human methaemoglobin by solvated electrons a non-equilibrium low-spin state of deoxyhaemoglobin is formed which has the characteristic haemochrome spectrum. This haemochrome state is ascribed to a weakly 6-coordinated structure of the haem, which is stabilised by the protonated distal histidine. Oxygen binding is not inhibited by the presence of the weak interaction in the haemochrome state. From the pH dependence of the biphasic behaviour of the oxygen binding a pK of about 8.8 is obtained which is ascribed to the deprotonation of the distal histidine which is in the proximity of a negative ion. A model is proposed to explain the complex spin-equilibria observed in methaemoglobin. The enthalpy of activation of the decay of the haemochrome state is about 53 kJ x mol(-1) and increases to 90 kJ x mol(-1) in the presence of 1 M methanol, indicating a strong interaction between methanol and haemoglobin. Around pH 8.4 the rate constant of the binding of oxygen to the haemochrome state is so high that it may well be diffusion controlled.

Reduction of ferricytochrome c, methemoglobin and metmyoglobin by hydroxyl and alcohol radicals.

We have studied the reaction of ferricytochrome c, methemoglobin and metmyoglobin with OH and alcohol radicals (methanol, ethanol, ethylene glycol and glycerol). These radicals can be divided into three groups: 1. The OH radicals which reduce the ferricytochrome c with a yield of (30 +/- 10)% and methemoglobin with a yield of (40 +/- 10)%. They do not reduce metmyoglobin. The reduction is not a normal bimolecular reaction but is most probably an intramolecular electron transfer of a protein radical. 2. Methanol and ethanol radicals which reduce all three hemoproteins with a yield of (100 +/- 5)%. This reduction is a normal bimolecular reaction. 3. Glycerol radicals which do not reduce the ferrihemoproteins under our experimental conditions. Ethylene glycol radicals do not reduce ferricytochrome c and metmyoglobin but they do reduce methemoglobin with a yield of (30 +/- 10)%.

The spin-state transition of the hemochrome non-equilibrium conformation in partially reduced human methemoglobin. A pulse-radiolysis study of aqueous-methanol solutions of methemoglobin.

The effect of external parameters on the relaxation process of the hemochrome-type non-equilibrium conformation in partially reduced methemoglobin has been investigated. The relaxation of the intermediate ferrous low-spin state to the high-spin equilibrium conformation of hemoglobin appears to be facilitated particularly by protons and phosphate ions. In addition to studying the spin-state transition in aquomethemoglobin we have also studied it in complexes of the heme group in methemoglobin with fluoride, azide and cyanide anions.

A tunnelling model to explain the reduction of ferricytochrome c by H and OH radicals.

The kinetics of the reaction of OH radicals with ferricytochrome c was studied in the time range 1 microsecond to 1 s by means of pulse radiolysis. The OH radicals reduce ferricytochrome c by 40% +/- 10%. The time course of the reduction is explained by a mechanism whereby a radical formed after hydrogen has been abstracted from the outer surface of the protein reduces the iron by electron tunnelling. We have calculated that the reducing electron in the radical is bound with an energy of at least 1.75 eV and that the frequency factor of the tunnelling process is v=10(11.5)s-1. This model accounts for the observed absorbance change in time range 5 . 10(-6)--10(-1)s. The time course of the reduction of ferricytochrome c by H radicals (Lichtin, N.N., Shafferman A. and Stein, G. (1974) Biochim. Biophys. Acta 357, 386--398) is explained by the same model.

Heterogeneity in the kinetics of oxygen binding to partially reduced human methemoglobin. A pulse-radiolysis study of oxygenated solutions of methemoglobin.

The pulse-radiolysis technique has been introduced because it permits a rapid reduction (in a few microseconds) of one heme group of the methemoglobin tetramer by hydrated electrons. The kinetics of the binding of oxygen to this particular valence intermediate (Hb3+) with one reduced alpha or beta subunit has been studied. It appears that the hydrated electrons preferentially reduce one type of subunit of methemoglobin at acid and neutral pH-values as is shown by the biphasic behaviour of Hb3+ on oxygenation. The second-order on-rate constants measured for the binding of oxygen to Hb3+ are 14 +/- 3 mM-1 ms-1 and 56 +/- 9 mM-1 ms-1, respectively. The relative contribution of the faster fraction is about 0.63 +/- 0.08 of the total oxygenation process. A comparison of the kinetic absorbance difference spectrum for the reduction of methemoglobin with the static difference spectrum of deoxyhemoglobin and methemoglobin in the Soret-region revealed a decreased absorbance of the unliganded subunit of Hb3+ at 430 nm. This fact suggests that Hb3+ is in the relaxed quaternary conformation, which is in agreement with the observed on-rate constants.