K.J.H. Van Buuren

Biochemical and biophysical studies on cytochrome c oxidase XIV. The reaction with cytochrome c as studied by pulse radiolysis

Abstract 1. The reduction of cytochrome c oxidase by hydrated electrons was studied in the absence and presence of cytochrome c . 2. Hydrated electrons do not readily reduce the heme of cytochrome c oxidase. This observation supports our previous conclusion that heme a is not directly exposed to the solvent. 3. In a mixture of cytochrome c and cytochrome c oxidase, cytochrome c is first reduced by hydrated electrons ( k = 4 · 10 10 M −1 · s −1 at 22 °C and pH 7.2) after which it transfers electrons to cytochrome c oxidase with a rate constant of 6 · 10 7 M −1 · s −1 at 22 °C and pH 7.2. 4. It was found that two equivalents of cytochrome c are oxidized initially per equivalent of heme a reduced, showing that one electron is accepted by a second electron acceptor, probably one of the copper atoms of cytochrome c oxidase. 5. After the initial reduction, redistribution of electrons takes place until an equilibrium is reached similar to that found in redox experiments of Tiesjema, R. H., Muijsers, A. O. and Van Gelder, B. F. (1973) Biochim. Biophys. Acta 305, 19–28.

Biochemical and biophysical studies on cytochrome aa3 I. Steady-state kinetics of cytochrome aa3

Abstract 1. 1. The steady-state kinetics of the reaction in the system: ascorbate → cytochrome c → cytochrome aa 3 → O 2 were studied, measuring both the degree of reduction of cytochrome c and the rates of O 2 consumption. 2. 2. In disagreement with some earlier reports it could be shown that the sole reaction of ascorbate is the reduction of ferricytochrome c . From the kinetics of the overall reaction, the rate constant for the reaction between ferricytochrome c and ascorbate could be calculated to be 23 M −1 ·sec −1 , in fair agreement with the value of 50 M −1 ·sec −1 obtained from direct stopped-flow measurements. 3. 3. Comparison of the rates of O 2 uptake at infinite cytochrome c and ascorbate concentrations for heart-muscle preparation and isolated cytochrome aa 3 revealed the presence of an inhibitor in the latter preparation. A mechanism of inhibition based on Minnaert s Mechanism IV , in which the inhibitor reacts with E , ES and EP forming inactive complexes with about the same K i , is in agreement with the experimental data. 4. 4. The K D for ES and EP , calculated from the kinetics of the overall reaction, was found to be 30 and 30–40 μM for the isolated and particulate cytochrome aa 3 , respectively. By making use of the data of Gibson et al. ( J. Biol. Chem. , 240 (1965) 888) on the reaction between ferrocytochrome c and ferricytochrome a , values for k −1 and k 2 of 1200 and 300 sec −1 , respectively, were calculated. 5. 5. Minnaert s Mechanism IV ( Biochim. Biophys. Acta , 50 (1961) 23) gives the simplest explanation of the observed steady-state kinetics.

Biochemical and biophysical studies on cytochrome c oxidase. XI. Reaction with azide

Abstract 1. Fluoride is a mixed-type inhibitor of the cytochrome c oxidase activity with a K i for the free enzyme of 10 mM and a K i for the cytochrome c -complexed enzyme of 35 mM. 2. Fluoride shifts the γ-band of the enzyme from 423 to 421 nm and the α-band from 597 to 598 nm. The difference spectrum (oxidized enzyme in the presence of fluoride minus oxidized enzyme) has peaks at 400, 453, 482, 605 and 638 nm and troughs at 430, 520, 552 and 674 nm. The changes in absorbance are small (about 3% at absorbance maxima) with respect to those of other hemoproteins. 3. On addition of fluoride to isolated cytochrome c oxidase 3 reactions can be distinguished: (I) a bimolecular binding reaction ( K on = 4 M −1 · s −1 and k off = 2.9 · 10 −2 s −1 at 25 °C, pH 7.4) contributing at 638 nm and 430 nm; (II) a first-order reaction ( k = 2.4 · 10 −2 ) s −1 at 22 °C, pH 7.2) visible mainly at 430 nm and (III) a very slow reaction with a half-time in the order of 10 min. 4. The spectroscopic dissociation constants for the fluoride binding, determined from Hill plots using the absorbance changes at 638 and 430 nm, are similar (7 and 10 mM, respectively, at 22 °C, pH 7.2). 5. A mechanism for the reaction is discussed in which the bimolecular binding reaction is followed by a conformational change of the enzyme-fluoride complex.

Purification and characterization of enkephalin-degradating enzymes from calf-brain striatum

Enkephalinase A and B are extracted from Triton-X 100 washed calf-brain particles and purified by DEAE-cellulose chromatography. Both enzymes have identical Km values in their membrane-bound and soluble form. Enkephalinase A has a pH optimum at 6.9 and a Km for Leu-enkephalin of 20-25 microM, which hardly depends on the pH. Thiorphan and phosphate are purely competitive inhibitors of Enkephalinase A with Ki values of 3 nM and 1.5 mM respectively (pH = 6.85). Enkephalinase B is not affected by phosphate or thiorphan. It has a Km for Leu-enkephalin of 10 microM, a pH optimum of 7.0 and is inhibited by low concentrations of apolar dipeptides.

Inhibitors of calf-brain enkephalinase A and B

Enkephalinase A and B isolated from calf-brain striatum have comparable substrate specificity (Km for Leu-enkephalin = 1-3.10(-5) microM) but a quite different affinity for certain inhibitors: phosphate, Secobarbital and Thiorphan are effective inhibitors for Enkephalinase A (IC50 of 2.5 mM, 30 microM and 4 nM respectively), while Enkephalinase B does not react with any of these compounds. Both enzymes are inhibited by 1 mM EDTA and o-phenanthroline indicating the presence of a metal atom in or near their active sites. Although with different abilities, both enzymes recognize dipeptides having at least one hydrophobic amino-acid side chain. The potency of such dipeptides can be used for a description of the active site.

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.

Hydrophobic properties of model compounds with peptide-like chemical environment: N-acetyl-N′-methyl-amino acid amides

Capacity factors in reversed-phase HPLC and distribution constants in octan-l-ol/water of N-acetyl-N′-methylamino acid amides have been measured as a function of temperature. The HPLC capacity factors are proposed as estimates of the hydrophobicity of the amino acid side chains.

Effect of various enkephalin analogs and dipeptides on the enzymatic activity of enkephalinase B isolated from calf-brain striatum

The inhibitory potency of several enkephalin analogs and dipeptides on the calf-brain enkephalinase B activity was established with the aim to characterize its active site. Highest potency was measured for dipeptides with a large side chain on both amino acids. The nature of the distal amino acid is of minor importance, provided it is not a glycine. Free carboxylic function is required for good interaction, whereas the stereochemical configuration of the dipeptide is less so. Enkephalinase B has only little affinity for D-Ala2-Leu-enkephalin. The data are to be used for the design of new enkephalinase B inhibitors.

Evidence for the presence of di- and triphospho pyridine nucleotide dehydrogenase derivatives as consistent contaminants of purified beef heart cytochrome-c oxidase.

Purified beef heart cytochrome-c oxidase preparations derived by three different laboratories contain NADH-K3 Fe(CN)6, NADH-nitrobluetetrazolium, and NADPH-nitrobluetetrazolium reductases. This is true of preparations exhibiting heme aa3 to protein ratios considered indicative of an excellent purity. An apparent association of cytochrome-c oxidase and one or more of the contaminants persists through immunodiffusion and nondenaturing electrophoresis and, in addition, in one instance copurification of NADH-K3 Fe(CN)6 reductase and cytochrome-c oxidase to a constant ratio of specific activities was demonstrated. Cytochrome-c oxidase can be freed of the contaminants by equilibration with an NAD+-affinity matrix. As a concomitant of equilibration with the matrix, theKM of cytochrome-c oxidase for ferrocytochrome-c is invariably decreased. Rate constants at low ferrocytochrome-c concentrations are consistently enhanced in all oxidase preparations upon equilibration with the NAD+ matrix. However, the effects of such equilibrations on the extrapolatedVmax varies from one preparation to another. Polyacrylamide gel electrophoresis in SDS-urea systems establishes that each of the preparations contains a minimum of three contaminants, each of an apparent formula weight of greater than 40,000 Daltons. NADH-NBT reductase was found to have a formula weight of approximately 46,000 Daltons. Their properties establish that NADH-K3 Fe(CN)6 and NADH-NBT reductases are separate proteins; the separate identity of NADPH-NBT reductase has not yet been determined.

Biochemical and biophysical studies on cytochrome aa3 IX. Reaction of cytochrome aa3 with hydrazine

Abstract 1. Aerobic incubation of purified cytochrome aa3 with hydrazine, even in less than stoichiometric amounts, produces one of the so called ‘oxygenated’ forms of the enzyme via an O2- intermediate. 2. The O2- ion is not a universal intermediate in the formation of ‘oxygenated’ cytochrome aa3. 3. Oxidation of hydrazine by cytochrome aa3 follows Michaelis-Menten kinetics with a Km of 3.8 mM and a maximum turnover number of 0.07 electron per mole cytochrome aa3 per s at pH 7.2 and 20 °C. 4. The reactions mentioned under 1 and 3 differ in kinetic pattern, Km for oxygen and sensitivity towards superoxide dismutase. 5. Hydrazine is a competitive inhibitor of cytochrome c oxidation by cytochrome aa3 with a Ki of 25 mM at pH 7.2 and 20 °C.