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Abstract— The Haber‐Weiss cycle: was investigated at low pH by radiolysis of oxygen or nitrogen saturated solutions of hydrogen peroxide. It was found that reaction 2 has a low rate constant: k2= 3.0 ± 0.6 M‐1 s‐1 (pH 2.3, 22°C). The rate determining step of reaction 2 is most probably the transfer of an electron from a π8* orbital of HO2 to the empty s̀u* orbital of H2O2. Overlap between these two orbitals is hindered by the filled π8* orbitals of H2O2. Fe(HI)EDTA catalyses reaction 2.

NADPH binding to p-hydroxybenzoate hydroxylase from Pseudornonas JZuorescens 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 > 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 monopoledipole 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.p-Hydroxybenzoate hydroxylase from Pseudornonas jluorescens is an external flavoprotein monooxygenase catalyzing the hydroxylation of p-hydroxybenzoate to 3,4-dihydroxybenzoate. The reaction requires NADPH as an electron donor [l]. It has been observed that the reduction of the proteinbound FAD by NADPH is accelerated enormously upon substrate binding [2]. This 'effector' role was shown to be a property of several substrate analogues, which are not necessarily transformed to a product [3]. The enhancement of the rate of reduction of FAD cannot be explained by an increase in NADPH binding since the dissociation constant of the enzyme-NADPH complex decreases only slightly at pH 6.6 and about 10-fold at pH 7.5 in the presence of the substrate or effector [I]. It has been suggested, for the enzyme from Pseudornonas desrnolytica, that the substrate p-hydroxybenzoate not only stimulates the rate of reduction of FAD by NADPH but also shifts the pH optimum of the binding of NADPH to more alkaline pH values [4]. This proposal is related to the fact that optimum binding of NADPH or NADH to the free enzyme occurs at about pH 6 whereas the enzyme possesses its optimal activity at a pH value of about 8. The data of the apparent pH-dependent dissociation constants for the complex between enzyme and NADPH or NADH Abbreviations. t-NADPH, 1,4,5,6-tetrahydronicotinamide adenine dinucleotide 2'-phosphate; Mes, 4-morpholineethanesulfonic acid; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid ; Hepps, 4-(2-hydroxyethyl)-l -piperazinepropanesulfonic acid ; 2',5'-ADP, adenosine 2',5'-bisphosphate.Enzyme. p-Hydroxybenzoate hydroxylase (EC 1.14.1 3.2). Definition. Debye, unit of dipole moment = 3.34 x 10-30 ml.served as further support for this proposal. They parallel those of the apparent pH-dependent rat...

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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 ( k , z los s-') the intermediate state decays to the stable semiquinone form of flavodoxin. The d C of the conformational change at pH 8 is about 29 kJ mol-(0.3 eV). This means that the upper limit for the pKof N-5 in the semiquinone form will be 13. The activation energy of the conformational change is 43 kJ mo1-l (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 i 1) x lo8 M-s-5) mV.(PH 8, I = 55 mM). The midpoint potential of the semiquinone/hydroquinone was found to be -(408 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 < Em (Fld/FldH -) < -290 mV. The exact value will depend on the AG 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.Flavodoxin (from Megasphaera elsdeizii) is a negatively charged protein with a molecular mass of about 16000 Da, which contains FMN as the prosthetic group. Flavodoxin is assumed to be an electron donor and acceptor for hydrogenase and is formed in great quantities if the bacteria are grown in an iron-deficient medium. When iron is present, ferredoxin is formed. So in the obligatory hydrogen production of the bactcrium flavodoxin takes over the role of ferredoxin. M . elsdenii ferredoxin contains two [4Fe-4S] clusters.The oxidation-reduction properties of flavodoxin can be described by two one-electron steps which have different midpoint potentials. At pH 8 the redox behaviour is given by Eqns (1) and (2) [l].where Fld is the oxidized form of flavodoxin, FldH the halfreduced or semiquinone form and FldH-the fully reduced or hydroquinone form. For more detailed information see the excellent review article of Mayhew and Ludwig [l]. As was proposed by Muller et al. [2], the semiquinone form is probably stabilized by the protonation of the N-5 of the FMN and/or the subsequent formation of a hydrogen bond between this proton and the peptide backbone. In the semiquinone form of flavodoxin from Clostridium M P there is probably a hydrogenAhhreviutions. MV'', methyl viologen; BV", benzyl viologen; Fld, flavodoxin; F...