Dan Bäckström

Electron Paramagnetic Resonance of the Copper in Dopamine beta-Monooxygenase. Rapid Reduction by Ascorbate, the Steady-State Redox Level, Chelation with EDTA, and Reactivation of the Apoenzyme by Added Copper

The water-soluble form of dopamine beta-monooxygenase from bovine adrenal medulla was studied. Addition of excess CuSO4 to purified enzyme preparations followed by extensive ultrafiltration against copper-free buffer at pH 7.0, gave preparations with about four copper atoms per enzyme tetramer of Mr 290 000. The enzyme-bound copper was shown by the rapid-freeze technique and electron paramagnetic resonance (EPR) to be reduced by ascorbate at a rate which was faster than the overall catalytic rate; about 10% of the copper was oxidized to Cu(II) during steady-state catalysis in the presence of excess ascorbate. These results support an electron-transfer function of the enzyme-bound copper during catalysis and indicate that the reduction by ascorbate is not the rate-limiting step. The enzyme-bound copper was rapidly chelated by EDTA at pH 7.0., and the apoenzyme thus obtained after dialysis revealed no EPR-detectable copper. Addition of CuSO4 to the apoenzyme gave an EPR spectrum similar to that of the native enzyme, and the apoenzyme was fully reactivated by the optimal concentration of CuSO4 in less than 2 s.

An iron sulfur protein in the mitochondrial outer membrane, reducible by NADH and NADPH

Abstract On addition of NADH or NADPH to the mitochondrial outer membrane fraction from rat liver, an electron paramagnetic resonance (EPR) spectrum is observed which is characteristic of a protein, containing an iron-sulfur center. The g-values are 2.01, 1.94 and 1.89. Quantitation of the EPR absorption and analysis of the acid labile sulfur content suggest that the paramagnetic center contains two iron and two acid labile sulfur atoms. The concentration of the center in the outer membrane is about 0.5 nmoles/mg protein.

Two iron—sulfur centers in mitochondrial outer membranes from beef heart as prepared by free‐flow electrophoresis

Mitochondrial outer membranes from rat liver were shown [I] to give rise to an EPR signal analogous to that of [2 Fe-2 S] centers, when NADH, NADPH or dithionite was added. As 2 Fe and 2 acidlabile S atoms were found per free spin, the presence of a [2 Fe-2 S] center in these outer membranes was postulated [l] . A similar EPR signal in preparations enriched in outer membranes from beef-heart mitochondria was found [2,3] . Recently a Fc-S protein with similar EPR propertics has been partially purified from beef-kidney cortex mitochondrial outer membranes [4] . On close inspection of the line shape of the EPR signal, we felt that the signal could not be due to a single S = % system. We report here the results of an EPR lineshape analysis using high-purity outer membranes from beef-heart mitochondria, which were isolated by means of free-flow electrophorcsis [5] . The isolation and the propcrtics of these membranes are also reported. The EPR line shape of the beefheart mitochondr-ial outer membranes was compared with the line shape of the partially purified Fe-S protein from the outer membranes of beef-kidney cortex mitochondria [4] .

Drug metabolism in isolated rat liver cells.

Although considerable knowledge has been gathered on the functional aspects of microsomal monooxygenation, comparatively little has so far been known about the intracellular regulation of this process. For such studies, we have found the isolated rat liver cell system to be a very useful model, combining the convenience of an in vitro system with the access to the complex mechanisms of the intact in vivo system. This model has the advantage over the perfused liver that it readily lends itself to the study of rapid reaction sequences and makes quantitation of short-term drug metabolic reactions easier. It is also superior to liver slices which often show considerable leakage of adenine and pyridine nucleotides and where substrate penetration and oxygen diffusion may present problems depending on the relative thickness of the slice.

The mechanism of oxidation of reduced nicotinamide dinucleotide phosphate by submitochondrial particles from beef heart.

1. Oxidation of NADPH by various acceptors catalyzed by submitochondrial particles and a partially purified NADH dehydrogenase from beef heart was investigated. Submitochondrial particles devoid of nicotinamide nucleotide transhydrogenase activity catalyze an oxidation of NADPH by oxygen. The partially purified NADH dehydrogenase prepared from these particles catalyzes an oxidation of NADPH by acetylpyridine-NAD. In both cases the rates of oxidation are about two orders of magnitude lower than those obtained with NADH as electron donor. 2. The kinetic characteristics of the NADPH oxidase reaction and reduction of acetylpyridine-NAD by NADPH are similar with regard to pH dependences and affinities for NADPH, indicating that both reactions involve the same binding site for NADPH. The binding of NADPH to this site appears to be rate limiting for the overall reactions. 3. At redox equilibrium NADPH and NADH reduce FMN and iron-sulphur center 1 of NADH dehydrogenase to the same extents. The rate of reduction of FMN by NADPH is at least two orders of magnitude lower than with NADH. 4. It is concluded that NADPH is a substrate of NADH dehydrogenase and that the nicotinamide nucleotide is oxidized by submitochondrial particles via the NADH--binding site of the enzyme.

Evidence for the presence of an iron sulphur protein in rat kidney cortex microsomes.

Abstract An electron paramagnetic resonance (EPR) study of the microsomal fraction isolated from rat kidney cortex has provided evidence for the presence of cytochrome P-450 in the low-spin form (g = 2.42, 2.25 and 1.91) but also revealed the appearance of an EPR signal (g = 2.01, 1.93 and 1.88) of increasing intensity upon reduction with NADPH or dithionite. The characteristics of this signal lead us to suggest that it is ascribable to an iron-sulphur protein presumably involved in the electron transfer from NADPH to cytochrome P-450.

Characterization of the iron-sulfur protein of the mitochondrial outer membrane partially purified from beef kidney cortex.

The iron-sulfur protein present in the mitochondrial outer membrane has been partially purified from beef kidney cortex mitochondria by means of selective solubilization followed by DEAE-cellulose chromatography. The EPR spectrum of the iron-sulfur protein with g-values at 2.01, 1.94 and 1.89 was well resolved up to 200 K which is unusual for an iron-sulfur protein. Analyses confirmed a center with two iron and two labile sulfur atoms in the protein. By measuring the effect of oxidation-reduction potential on the EPR signal amplitude, midpoint potentials at pH 7.2 were determined both for the purified iron-sulfur protein, +75 (+/- 5) mV, and in prepared mitochondrial outer membrane, +62 (+/- 6) mV. At pH 8.2 slightly lower values were indicated, +62 and 52 mV, respectively. The oxidation-reduction equilibrium involved a one electron transfer. A functional relationship to the rotenone-insensitive NADH-cytochrome c oxidoreductase in the mitochondrial outer membrane is suggested. Both this activity and the iron-sulfur center were sensitive to acidities slightly below pH 7 in contrast to the iron-sulfur centers of the inner membrane.

Formation of microsomal cytochrome P-450 complexes studied by the NMR relaxation of water☆

Cytochrome P-450 in microsomes from liver of phenobarbital treated and control rats has been studied by light absorption and by magnetic resonance methods (EPR and NMR). The nuclear relaxation rate of water protons was measured for microsomal suspensions in the presence of various reactants of Type I and II. The change of relaxation rates correlates well with the spin state conversion of the heme iron. No competition between eventual inner-sphere water molecules and the reactants seems to occur. The temperature dependence of the low spin to high spin equilibrium was studied by light absorption and was accounted for in the temperature variation of the molar relaxation rates of the two spin states.