Biomedical subjects
Publications and source records attributed to M Brunori.
Temperature-jump studies on the electron-transfer reaction between azurin and cytochrome c-551 clearly reveal two chemical relaxations. The amplitudes of these relaxation processes have identical spectral distributions, but the relaxation times show different dependences on the reactant concentrations. These findings are discussed in terms of possible models.
A number of methods were used to prepare a species of mammalian cytochrome oxidase (EC 1.9.3.1, ferrocytochrome c-oxygen oxidoreductase) in which only cytochrome a(3) is reduced and in combination with CO. The kinetics of CO binding by cytochrome a(3) (2+) in this species is significantly different from that exhibited by cytochrome a(3) (2+) in the fully reduced enzyme. The second-order rate constant for combination was 5x10(4)m(-1).s(-1) and the ;off' constant was 3x10(-2)s(-1). The kinetic difference spectra cytochrome a(3) (2+)-cytochrome a(3) (2+)-CO reveal further differences between the mixed-valence and the fully reduced enzyme. The reaction between cytochrome a(3) (2+) and oxygen in the mixed-valence species was followed in flow-flash experiments and reveals a fast, oxygen-dependent (8x10(7)m(-1).s(-1) at low oxygen) rate followed by a slow process, whose rate is independent of oxygen but whose amplitude is dependent on [O(2)]. The fast oxygen-dependent reaction yields as the first product the so-called ;oxygenated' enzyme. We conclude from these experiments that the ligand-binding behaviour of cytochrome a(3) depends on the redox state of its partners, a fact which represents clear evidence for site-site interaction in this enzyme. The fact that oxygen reacts rapidly with this enzyme species in which only one component, namely cytochrome a(3), is reduced represents clear and unequivocal evidence that this is indeed the O(2)-binding site in cytochrome oxidase and may indicate that reduction of oxygen can proceed via single electron steps.
1. The decay of oxyferroperoxidase to ferriperoxidase was studied by rapidly mixing solutions of ferroperoxidase with various amounts of oxygen and following the time-course of appearance of oxyferroperoxidase and its subsequent decay to ferriperoxidase by reaction with ferroperoxidase. 2. The scheme can be accommodated by and [Formula: see text] and occurs without detectable intermediates being observed in the millisecond time-range. 3. Analogue-computer simulation of the reaction is in agreement with the initial rate-limiting reaction being an intermolecular, not intramolecular, electron-donating process, and analysis of the data leads to quantitative values for the rate constants of the overall process. 4. The reaction of oxyferroperoxidase with ferroperoxidase is a model of a terminal oxidase, and the results are discussed in terms of the possible importance of this reaction in peroxidase function, and also in the light it throws on autoxidation of haem compounds.
Studies on horse heart cytochrome c polymers were carried out by stopped-flow and photolysis techniques, to investigate the properties of the CO complex and the kinetics of electron transfer, mainly of the dimeric and tetrameric forms. CO binding, which does not occur with native monomers, proceeds at both pH7.0 and pH9.6, and appears to follow complex kinetics: an initial phase is observed, which is CO-concentration-dependent, followed by a very slow monomolecular phase (k~2x10(-3)s(-1) at pH7) before establishment of equilibrium. Photodissociation of the CO complex has a very low quantum yield, probably less than 0.1. Static titration data of the dimer gave an ;n' value of 0.4. These data strongly suggest heterogeneity of the population of binding sites, and have been interpreted in terms of the existence of different structures, probably owing to the non-unique type of binding of monomers during polymerization. Polymers of cytochrome c carboxymethylated on the methionine residue normally ligated to iron show simple CO recombination kinetics after photolytic removal (k(on)=1.5x10(6)m(-1).s(-1) at pH6). We therefore suggest that, for native cytochrome c, polymerization has an effect on the lability of the haem crevice, rendering the iron available for binding ligands, without, however, forming the structure of a truly open crevice. Electron transfer is, on the other hand, a simple process, and no gross differences are observed between monomer and polymers. A simple model, taking into account all these data, is suggested.
The binding of nitric oxide to hemoglobin components of trout (Salmo irideus), i.e., Hb trout I and Hb trout IV, has been studied by optical and electron paramagnetic resonance spectroscopy. Kinetic studies show that the Root effect in Hb trout IV is operative also for NO, since a large increase in the dissociation velocity constant (j(4)) is observed as the pH is decreased below 7. Moreover, the time course of the displacement of NO by CO is heterogeneous, suggesting that alpha and beta chains may have different j(4) values. Low-temperature X-band electron paramagnetic resonance spectra have been recorded with Hb trout I and IV saturated with NO at different pH values. The spectra of Hb trout IV are strongly pH-dependent. The high-pH form (pH 8.1) shows axial symmetry and no resolved hyperfine splitting, while the low-pH form is rhombic with a hyperfine splitting of 6.5 G in the g(z) region. The latter form reflects a more distorted site with a more significant delocalization of the unpaired electron on the proximal histidine; both features indicate a destabilization of the ligand binding at low pH. On the other hand, spectra of Hb trout I are axial at both pH values, with hyperfine splitting of 16.5 G, indicating that the site is not distorted and interacts with the ligand very strongly at either pH.
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The quantum yield, [unk], is reported for the photodissociation of CO from reduced carboxymethylated cytochrome c. The values of [unk] obtained are low relative to that for myoglobin and are pH-independent, being 0.23 at pH6.1 and 0.27 at pH9.7.
The kinetics of the reaction between O(2) and haemoglobin bound to haptoglobin 1-1 were investigated by the stopped-flow and temperature-jump techniques. The reaction, which follows second-order kinetics in the lower concentration range, becomes independent of O(2) concentration above about 150mum-O(2).
We report results of the application of the "pulse method" to the study of photodissociation of various ligands from several heme proteins. By use of this technique, which allows the determination of the quantum yield of photodissociation accurately and rapidly, several ligands (CO, O(2), isocyanides) have been investigated for sperm-whale myoglobin and trout hemoglobin I. In agreement with previous results, the new data lead to the conclusion that no simple relationship exists between the quantum yield and the affinity constant in the ground state. For trout hemoglobin I, the experiments were extended to measure the quantum yield of the CO photodissociation as a function of the initial degree of saturation, from fully saturated down to the initial values of about 1.5%. The results yield additional information to that obtained by the "steady-state" method, and in particular exclude the idea that the photochemical yield is in any way dependent on the fractional saturation of the molecule with carbon monoxide.
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