Johan W. van Leeuwen

THE HABER‐WEISS CYCLE

Abstract— The Haber‐Weiss cycle:

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.

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.

Direct electron transfer between a chemically, viologen-modified glassy-carbon electrode and ferredoxins from spinach and Megasphaera elsdenii

Direct reversible electron transfer between an electrode and ferredoxins has been described by several authors [l-8]. Recently, we have shown that at the dropping mercury electrode the efficiency of reduction of both 4Fe-4FS and 2Fe-2S ferredoxins can be enhanced drastically [8]. This increase in reduction efficiency was obtained by addition of a positively charged polymer or surfactant to the negatively charged ferredoxins, indicating that electrostatic effects play an important, if not a predominating role. Authors in [5,6] have reported on the reduction and oxidation of spinach ferredoxin at a gold electrode on which a polymeric form of methyl viologen was adsorbed. Authors in [7] have described the direct reversible

A pulse radiolysis study of electron tunneling in an 8 M NaOH glass between 4 and 100 K

Pulse radiolysis experiments have been performed in an 8 M NaOH glass between 4 and 100 K and in the time range 10−6 to 10 s. The spur radius was estimated from the decay of the trapped electrons on the red side of the absorption maximum. The spur radius was about 4 nm at 80 K and increased to about 6 nm at 10 K. We studied the decay of trapped electrons in the presence of the following electron scavengers: CrO2−4, Fe(CN)3−6, and NO−2. No temperature dependence was found between 10 and 100 K. To explain the results distance‐dependent and/or time‐dependent Franck–Condon factors have been introduced. We show that in the time range studied it is not possible to distinguish between direct tunneling and trap‐to‐trap tunneling. Dry electron scavenging efficiencies and encounter pair formation are expressed in S37. For CrO2−4, Fe(CN)3−6, and NO−2 we found that S37 was 45, 130, and 300 M, respectively.

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)%.

Pulse-radiolytic studies on the spin-state transitions in aquomethemoglobin after reduction of a single heme group

We have previously observed a transient state (halftime ‘L 15 ps) in aquomethemoglobin with an absorption maximum near 420 nm after rapid reduction of a single ferric heme group by hydrated electrons [l] . This observation has been confirmed [2,3] . Moreover, this microsecond process disappeared in the presence of Ins-P6 [l] . On binding this organic phosphate, metHb changes its quaternary conformation from the Rto the T-state [4]. Therefore, we concluded that in the absence of an allosteric effector the reduction reaction of one heme group in metHb by hydrated electrons was followed by a quaternary conformational change (R + T transition). spin-state. The subsequent transition of the ferrous low spin to the stable ferrous high spin-state proceeds with a half-time that depends on the solvent condition. On the basis of these results we have postulated a two-step reduction mechanism for aquomethemoglobin.

A study of the temperature dependence of the visible absorption band of trapped electrons in ethylene glycol-water and LiC1 glasses between 4 and 100 K

Abstract Spectra of electrons trapped in ethylene glycol-water and in 8.5 M LiC1 glasses after pulse radiolysis have been measured between 4 and 100 K. In (50/50) ethylene glycol-water and in 8.5 M LiC1 glasses the yield of deep-trapped electrons (e - vis decreases by approximately a factor of three when the temperature is lowered from 100 to 4 K. This decrease is ascribed to an increase in the formation of shallow-trapped electrons (e - IR , λ max ≧ 3 μm). Hardly any e - IR is converted to e - vis . The growth observed in the yield of e - vis after the pulse is ascribed to deep trap relaxation. In the (50/50) ethylene glycol-water glass at 4 K the presence of large amounts of salts (≧ 2 M) causes an increase in the yield of e - vis . In the (70/30) ethylene glycol-water glass hardly any e - IR will form. A model is proposed in which an electron trapped in a water dimer is considered to be responsible for the IR absorption (λ max ≧3 μm).

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 > 5 SOD has a net negative charge.

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.