The reduction of the Rieske iron-sulfur cluster in naphthalene dioxygenase by X-rays.

Naphthalene 1,2 dioxygenase (NDO) displays characteristic UV-Vis spectra depending on the oxidation state of the Rieske center. Investigations on crystals of NDO grown for X-ray diffraction experiments showed spectra characteristic of the oxidized form. Crystals reduced in an anaerobic glovebox using sodium-dithionite showed a characteristic reduced spectrum. Spectra of crystals (cooled to 100 K) after being exposed to X-rays for data collection showed spectra corresponding to a reduced Rieske iron center, demonstrating the ability of X-rays to change the oxidation state of the Rieske iron-sulfur cluster in NDO.

EPR properties of mixed-valent mu-oxo and mu-hydroxo dinuclear iron complexes produced by radiolytic reduction at 77 K.

Radiolytic reduction at 77 K of oxo/hydroxo-bridged dinuclear iron(III) complexes in frozen solutions forms kinetically stabilized, mixed-valent species in high yields that model the mixed-valent sites of non-heme, diiron proteins. The mixed-valent species trapped at 77 K retain ligation geometry similar to the initial diferric clusters. The shapes of the mixed-valent EPR signals depend strongly on the bridging ligands. Spectra of the Fe(II)OFe(III) species reveal an S = 1/2 ground state with small g-anisotropy as characterized by the uniaxial component (gz-gav/2 < 0.03) observable at temperatures as high as approximately 100 K. In contrast, hydroxo-bridged mixed-valent species are characterized by large g-anisotropy (gz-gav/2 > 0.03) and are observable only below 30 K. Annealing at higher temperatures causes structural relaxation and changes in the EPR characteristics. EPR spectral properties allow the oxo- and hydroxo-bridged, mixed-valent diiron centers to be distinguished from each other and can help characterize the structure of mixed-valent centers in proteins.

Radiolytic reduction of methane monooxygenase dinuclear iron cluster at 77 K. EPR evidence for conformational change upon reduction or binding of component B to the diferric state.

The soluble form of methane monooxygenase (MMO) consists of three components: reductase, hydroxylase (MMOH), and "B" (MMOB). Resting MMOH contains a diferric bis-mu-hydroxodinuclear iron "diamond core" cluster which is the site of oxygen activation chemistry after reduction. Here it is shown that gamma-irradiation of MMOH at 77 K results in reduction of the diiron cluster to an EPR active Fe(II). Fe(III) mixed valence state. At this temperature, the conformation of the enzyme remains essentially unchanged during reduction, so the EPR-spectrum becomes a probe of the conformation of the diferric state. The gamma-irradiated MMOH exhibits EPR spectra that differ in lineshape and saturation properties from those of the mixed valence MMOH generated by chemical reduction in solution; annealing the gamma-irradiated sample at 230 K yields the spectra of the chemically reduced sample. This demonstrates that the conformation of diferric MMOH in the vicinity of the diiron cluster changes during reduction to the mixed valence state. The analogous experiment for the MMOB.MMOH complex gives a new mixed valence species following gamma-irradiation that differs from all previously reported mixed valence species. Thus, binding of MMOB also causes a change in the conformation of diferric MMOH. It is hypothesized that the structural changes observed for the first time here may involve conversion of the dihydroxo-bridged diamond core structure to one with more readily dissociable bridging oxygen ligands to facilitate reaction with O2 following cluster reduction.

[The effect of x-ray irradiation on the cytochrome P-450-dependent mono-oxygenase system of the liver in different animal species]. / Vliianie rentgenovskogo oblucheniia na tsitokhrom P-450-zavisimuiu monooksigenaznuiu sistemu pecheni u razlichnykh vidov zhivotnykh.

The whole X-irradiation (7 Gy) of male rat, mouse and guinea-pig caused in general similar alterations in the content of cytochrome P-450 and aminopyrine-N-demethylase activity in liver microsomes. On the 5-7th day after irradiation the parameters were 39-79% of the normal level. The same postradiation changes were observed in females of these animal species but in females of rats and guinea-pigs the effect was less expressed. The depression of activity in liver microsomal cytochrome P-450-dependent monooxygenase system has been concluded to be one of the characteristic features of acute form in radiation damage.

[Activity of the enzymatic drug-metabolizing system of the liver in combined radiation-thermal injury]. / Izuchenie aktivnosti fermentativnoi sistemy pecheni, metaboliziruiushchei lekarstvennye veshchestva, pri kombinirovannom radiatsionno-termicheskom porazhenii.

The functional status of the enzyme monooxygenase system of rat liver was assessed by the hexenal test at different time intervals following irradiation (6 Gy), thermal affection, and the combined effect of the two factors. The data obtained were indicative of the potentiation of the inhibiting action of the two factors on the detoxicating function of liver.

The activation of the cytochrome P-450 dependent monooxygenase system by light.

The cytochrome P-450 dependent monooxygenase system of rat liver microsomes is investigated by light dosimetry (action spectroscopy). The scoparone-O-demethylation activity is enhanced by light and depends on the wavelength of the irradiating light. The relative increase of the activity (about 15%) by the irradiating light (approximately 0.5 mW/cm2) is maximal at a wavelength of 400 nm. The light induced enhancement of the 7-ethoxycoumarin-O-deethylase activity was measured in reconstituted systems, consisting of one of two P-450 enzymes (P-450 beta NF-B and P-450 PB-B) with the NADPH-cytochrome P-450 reductase. The action spectrum of the reconstituted P-450 beta NF-B:NADPH-P-450 reductase complex shows a maximum between 420 and 440 nm. The relative increase of the activity induced by light of 420 nm was 7.3% and 9% for the reconstituted systems of P-450 beta NF-B and P-450 PB-B, respectively. The results are discussed in analogy to the classic experiments of Warburg with its blocked CO-enzyme-complex. The results can best be explained by the assumption that the light induced enhancement of the enzyme activity is due to an excitement of those intermediate states of the P-450 catalytic cycle (ferric and ferrous state of the heme iron) which are rate limiting.

Spectroscopic evidence for a photosensitive oxygenated state of ammonia mono-oxygenase.

Photoinactivation of ammonia oxidation by Nitrosomonas europaea cells by near-u.v. light was confirmed and further shown to occur with the same rate constant as loss of bromoethane-oxidation activity. Hydroxylamine oxidation was much less photosensitive. Protection against inactivation was afforded by anaerobiosis, organic substrates of ammonia mono-oxygenase such as bromoethane, or metal-ion-chelating agents such as thiourea. The presence of 10 mM-NH4+ or 1 mM-hydroxylamine made little difference, whereas hydrazine had a potentiating effect. Illumination of cells also caused a bleaching in the absorption spectrum around 380 nm, along with changes in the cytochrome gamma-band region. Similar effects below 400 nm were obtained when organic substrates and inhibitors of the mono-oxygenase were added to cells in the dark. The copper proteins haemocyanin and tyrosinase have a photosensitive oxygenated state with a near-u.v. absorption band of similar half-width. They also have a sensitivity to chelating agents similar to that of ammonia mono-oxygenase. The experimental results are explained in terms of a three-stage catalytic cycle analogous to that for tyrosinase. In resting cells most of the enzyme is believed to be in an oxygenated (Oxy) form, which absorbs maximally at 378 nm and is photosensitive. In the presence of a substrate, one O atom is inserted into the substrate and the other is reduced to water, leaving the enzyme in an oxidized (Met) state. This is followed by a two-electron reduction of the proposed binuclear copper site to give a reduced (Deoxy) state, which can bind O2 to complete the cycle.

Enzymic synthesis of juvenile hormone in locust corpora allata: evidence for a microsomal cytochrome P-450 linked methyl farnesoate epoxidase.

Homogenates of corpora allata from adult Locusta migratoria in phosphate-buffered EDTA have been analysed by sucrose-density-gradient centrifugation. Succinate-cytochrome c reductase activity (mitochondrial) bands between d20/4 1.13-1.15, whereas NADPH-cytochrome c reductase and NADPH-dependent methyl farnesoate 10.11-epoxidase activities band identically between d20/4 1.06-1.12. We conclude that the methyl farnesoate epoxidase is exclusively microsomal. Farnesoic acid O-methyltransferase is an exclusively soluble enzyme which stoichiometrically transfers the S-methyl group from S-adenosylmethionine to farnesoic acid. No carboxyl esterase activity was found. Isolated microsomes were used to obtain an apparent Km = 7.7 X 10-6 M for the epoxidase, although substrate solubility limits the rate to 0.5 V. As expected, the product (juvenile hormone III) is chiral (10 R). The epoxidase is inhibited by excess NADP+ and oxidised cytochrome c, but neither inhibited nor synergised by NADH. NADH supports less than 10% of the NADPH rate of epoxidation. The epoxidase is inhibited by a carbon monoxide/oxygen atmosphere, half-maximal inhibition occurring at a CO/O2 ratio of 4.0. This inhibition is reversed by white-light irradiation.