Torbjørn Ljones

Studies on the binding of copper to dopamine β-monooxygenase and other proteins using the Cu2+ ion-selective electrode

The binding of Cu2+ to native and copper-free dopamine beta-monooxygenase has been investigated by potentiometric titrations using a Cu2+-selective electrode. Stoichiometric formation constants have been determined from regression analysis of the resulting titration curves. The results establish a stoichiometry of four high-affinity binding sites for Cu/+ (log Kf approximately 11) per enzyme tetramer, and more binding sites of lower affinity (log Kf approximately 5-7). The data for binding of the first four Cu2+ to the enzyme tetramer indicate interactions in the binding to the sites. Bovine serum albumin, metal-free carbonic anhydrase, and ovotransferrin have also been titrated with Cu2+, and the formation constants of both high-affinity binding sites and other sites have been determined. The stoichiometry of one high-affinity binding site of Cu2+ for carbonic anhydrase (log Kf approximately 10-12) and two sites for ovotransferrin (log Kf approximately 11) agree with the reported metal binding properties of these proteins. The number of high-affinity binding sites for bovine serum albumin was pH dependent.

A continuous-wave electron-nuclear double resonance (X-band) study of the Cu2+ sites of particulate methane mono-oxygenase of Methylococcus capsulatus (strain M) in membrane and pure dopamine beta-mono-oxygenase of the adrenal medulla.

All methanotrophic bacteria express a membrane-bound (particulate) methane mono-oxygenase (pMMO). In the present study, we have investigated pMMO in membrane fragments from Methylococcus capsulatus (strain M). pMMO contains a typical type-2 Cu(2+) centre with the following EPR parameters: g(z) 2.24, g(x,y) 2.06, A(Cu)(z) 19.0 mT and A(Cu)(x,y) 1.0 mT. Simulation of the Cu(2+) spectrum yielded a best match by using four equivalent nitrogens (A(N)=1.5 mT, 42 MHz). Incubation with ferricyanide neither changed nor increased the amount of EPR-active Cu(2+), in contrast with other reports. The EPR visible copper seems not to be part of any cluster, as judged from the microwave power saturation behaviour. Continuous-wave electron-nuclear double resonance (CW ENDOR; 9.4 GHz, 5-20 K) experiments at g( perpendicular) of the Cu(II) spectrum show a weak coupling to protons with an A(H) of 2.9 MHz that corresponds to a distance of 3.8 A (1 A identical with 0.1 nm), assuming that it is a purely dipolar coupling. Incubation in (2)H(2)O leads to a significant decrease in these (1)H-ENDOR intensities, showing that these protons are exchangeable. This result strongly suggests that the EPR visible copper site of pMMO is accessible to solvent, which was confirmed by the chelation of the Cu(2+) by diethyldithiocarbamic acid. The (1)H and (14)N hyperfine coupling constants confirm a histidine ligation of the EPR visible copper site in pMMO. The hyperfine structure in the ENDOR or EPR spectra of pMMO is not influenced by the inhibitors azide, cyanide or ammonia, indicating that they do not bind to the EPR visible copper. We compared pMMO with the type-2 Cu(2+) enzyme, dopamine beta-mono-oxygenase (DbetaM). For DbetaM, it is assumed that the copper site is solvent-accessible. CW ENDOR shows similar weakly coupled and (2)H(2)O-exchangeable protons (2.9 MHz), as observed in pMMO, as well as the strongly coupled nitrogens (40 MHz) from the co-ordinating N of the histidines in DbetaM. In conclusion, the resting EPR visible Cu in pMMO is not part of a trinuclear cluster, as has been suggested previously.

Proton n.m.r. of ferrodoxin from Clostridium pasteurianum

Proton magnetic resonance spectra at 500 MHz are reported for the oxidized and reduced forms of the 2[4Fe-4S]-ferredoxin from Clostridium pasteurianum. The reduced protein showed additional peaks in the 10-60 ppm region, which were previously unobserved, and there were significant differences between oxidized and reduced states in the whole region. The electron exchange rate in partially reduced ferredoxin is slow on the n.m.r. time scale when reduced with sodium dithionite, but fast when zinc reduced methyl viologen is used as reducing agent. We explain the difference between fast and slow exchange as being due to the different chemical properties of the two reducing agents.

Ferredoxin from clostridium pasteurianum: Preparation and properties of the cobalt derivative

Abstract The two native (4Fe-4S)-clusters of ferredoxin from Clostridium pasteurianum were replaced with cobalt to give ferredoxin containing 2 atoms of cobalt per protein molecule. The cobalt derivative had optical absorption maxima at 270 nm (ϵ = 20,000 M−1 cm−1), 400 nm(ϵ = 12,100 M−1 cm−1),474nm (ϵ = 11,000 M−1 cm−1) and a shoulder around 600 nm. The visible spectrum of the Co-substituted protein was stable in the pH region from 4.8 to 10 at 4°C for hours and did not change when stored aerobically at 4°C at pH 7.5 for several days. There were no EPR signals at 77 K even after addition of excess CN−. The titration of sulfhydryl groups with PMPS revealed that there were no free titratable cysteine residues, while in native ferredoxin 24 mole PMPS react per mole protein. Reduction of the cobalt substituted protein with sodium dithionite gave a decrease in absorbance of approximately 30% of the initial value. The cobalt was not removed from the protein by EDTA.

Fluorescence studies on dopamine β-monooxygenase: effects of salts, pH changes, metal-chelating agents and Cu2+

The intrinsic protein fluorescence of dopamine beta-monooxygenase (3,4-dihydroxyphenethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) has been characterized. The fluorescence is dominated by emission from tryptophans in a hydrophobic environment. Changes in the conformation of the enzyme induced by anions, pH changes, metal-chelating agents and Cu2+ have been determined. Conformational transitions induced by anions take place at concentrations between 0.05 and 0.2 M. Most anions give rise to a blue-shift, while ClO4- induces a red-shift of the emission spectrum. pH dependence of the protein fluorescence revealed a conformational change between pH 6.0 and 5.0. The interactions between dopamine beta-monooxygenase and seven different metal-chelating agents have been investigated using protein fluorescence, heat inactivation, and inhibition measurements. All the metal-chelating agents are able to remove the active-site copper as demonstrated by complete inhibition of enzyme activity, restoration of activity by the addition of copper, and the observation that the enzyme becomes more sensitive to heat inactivation in the presence of chelating agents, thus behaving similarly to the copper-free apoenzyme. The charge and size of the chelating agents are of importance for the reaction with the active-site copper, which is consistent with a mechanism for removal of the copper, including a ternary enzyme-copper chelating agent complex. By contrast, under turnover conditions in the presence of substrates, dissociation of the active-site copper and chelation of the free copper is a dominating mechanism. Three distinct conformations were characterized on the basis of the fluorescence spectra and the degree of quenching by Cu2+ and I-. For the copper-free apoenzyme a unique binding site for binding of the first copper was demonstrated by larger quenching of the protein fluorescence than for binding of additional copper.

Oxygen disruption of the 2[4Fe-4S] clusters in Clostridium pasteurianum ferredoxin shown by 1H-NMR.

The ferredoxin from Clostridium pasteurianum, which contains two [4Fe-4S] clusters, was investigated in its oxidized and reduced states by two-dimensional (2D) (1)H-(1)H nuclear Overhauser enhancement spectroscopy (NOESY). Comparison of the data from the oxidized ferredoxin with those published previously revealed the same NOE connectivities. No previous (1)H-(1)H NOESY study of the fully reduced ferredoxin has previously been published. However, it was possible to compare our results with those of a 2D exchange spectroscopy investigation of half-reduced C. pasteurianum ferredoxin. The present results with reduced C. pasteurianum ferredoxin confirm many of the (1)H peaks and NOE interactions reported earlier, revise others, and locate resonances previously undetected. When the ferredoxin was slightly exposed to oxygen, several of the hyperfine shifted resonances were irreversibly influenced. A resonance at 34 ppm in the (1)H NMR spectra of both redox states is indicative of oxygen exposure. These results indicate the importance of keeping the ferredoxin strictly anaerobic during purification and solvent exchange.

Spectroscopic studies of cobalt-substituted ferredoxin fromClostridium pasteurianum

SummaryFerredoxin fromClostridium pasteurianum substituted with two Co atoms did not give any cobalt EPR signal at 8 K as isolated, but upon reduction with sodium dithionite, a broad signal appeared withg values that indicate highspin (S=3/2) Co(II). These signals were distinct from Co(II)-dithiothreitol signals, and disappeared upon reoxidation with air. Under anaerobic incubation of apoferredoxin with Co(II), a green derivative showed a visible spectrum typical of tetrahedral Co(Il)-thiolate coordination, which shifted dramatically upon exposure to air. The1H-NMR spectrum of the aerobically isolated protein is reported at 300 MHz; magnetic susceptibility measurements were indicative of a diamagnetic species. These spectroscopic studies indicate that Co(II)-substituted ferredoxin is oxidized to low-spin Co(III)-ferredoxin in the presence of sulfide and oxygen. The diamagnetic Co(III) state could reversibly be reduced to highspin Co(II) by sodium dithionite.

Enzymatic cleavage of Clostridium pasteurianum apoferredoxin and reconstitution of the cleaved products

Native ferredoxin from Clostridium pasteurianum proved to be resistant to proteolytic cleavage under anaerobic conditions, but was digested in the presence of air. Apoferredoxin was hydrolyzed by the proteinases used, while cobalt-substituted ferredoxin was resistant both under anaerobic and aerobic conditions. These studies indicate that metal binding of the protein stabilizes the folded state, which is extremely resistant to proteolytic attack. Sulfitolyzed apoferredoxin was subjected to specific cleavage by pepsin at pH 3.2, yielding two fragments. The fragments could be reconstituted to an unstable holoprotein with UV-visible absorption features like that of the native form.

The copper of dopamine β-Monooxygenase: High accessibility and rapid exchange

Abstract Dopamine β-monooxygenase (dopamine β-hydroxylase: EC 1.14.17.1) catalyzes the reaction: Dopamine + ascorbate + O2 → noradrenaline + dehydroascorbate + H2O (RH + 2e− + 2H+ + O2 → ROH + H2O) The purified water-soluble enzyme from bovine adrenal medulla contains 4 copper atoms per enzyme tetramer of 290,000 daltons. These copper atoms are essential for enzymic activity, and they most probably participate in both electron transfer and binding of O2 [1]. The copper in dopamine β-monooxygenase can be classified as type 2 copper according to its EPR spectrum, with a large hyperfine splitting and the low absorption in the visible spectrum (ϵ = 40 M−1Cu · cm−1 at 680 nm, the maximum of the Cu(II)-band) [1]. This enzyme-bound copper is, however, different from type 2 copper in the blue oxidases by other criteria, especially by showing a high accessibility of the copper sites. Thus, we have shown that the copper atoms of dopamine β-monooxygenase can be rapidly removed by chelators at nondenaturing conditions both in the reduced and oxidized states, and the inactive apoenzyme is reactivated in less than 2s by addition of CuSO4 [2, 3]. We have now studied the binding of 64Cu to this enzyme in reconstitution and exchange experiments. High performance size-exclusion gel chromatography with the protein analysis column I-125 from Waters was used to separate the enzyme-bound and free 64Cu, and the amount of 64Cu bound to the protein was determined from the radioactivity eluting together with the protein. Experiments with binding of 64Cu(II) to the apoenzyme give further evidence for a specific binding of 4 copper atoms per tetramer, but some weaker copper-binding sites were observed in the presence of an excess of copper. When the apoenzyme was incubated with 4 atoms of 64Cu(II) per tetramer, about 3.5 copper atoms were eluted with the protein indicating that the binding of Cu(II) is not extremely tight. Similar amounts of 64Cu were bound to the apoenzyme in the presence of ascorbate indicating the binding of Cu(II) is similar to that of Cu(II). The exchanges of both Cu(I) and Cu(II) in the holoenzyme are rapid and a half-life of about 1 min was estimated for the exchange of the enzyme bound Cu(II) in the presence of a two-fold excess of 64Cu(II) at pH 6.1. Experiments in the presence of ascorbate revealed that the exchange of Cu(I) was complete in 1 min at similar conditions. The exchange of the copper atoms in dopamine β-monooxygenase are thus much more rapid than reported for other copper proteins, and the present results point to unique copper-binding site in this protein.