Comparison of physico-chemical characteristics of four laccases from different basidiomycetes.

New strains of basidiomycetes producing extracellular laccases (Trametes ochracea 92-78, and Trametes hirsuta 56) have been found by screening of isolates of Trametes fungi. The laccases from T. hirsuta 56 and T. ochracea 92-78 as well as two laccases from previously found and described strains of basidiomycetes, namely Cerrena maxima and Coriolopsis fulvocinerea, were purified to homogeneity. The standard redox potentials of type 1 copper in the enzymes were determined and found to be 780, 790, 750, and 780 mV, respectively. The spectral and biochemical studies showed that the enzymes had no significant differences between the structures of their active sites (T1, T2, and T3). In spite of this fact, the basic biochemical properties as well as the redox potentials of the T1 sites of the enzymes were found to be different. The molecular weights of the laccases range from 64 to 70 kDa, and their pI values range from 3.5 to 4.7. The pH-optima are in the range 3.5-5.2. The temperature optimum for activity is about 50 degrees C. The thermal stabilities of the enzymes were studied. The catalytic and Michaelis constants for catechol, guaiacol, hydroquinone, sinapinic acid, and K(4)Fe(CN)(6) were determined. Based on these results as well as results obtained by comparing with published properties of several laccases, it could be concluded that T. hirsuta and Cerrena maxima laccases have some superior characteristics such as high stability, high activity, and low carbohydrate content, making them attractive objects for further investigations as well as for application in different areas of biotechnology.

Structure-function studies of iron-sulfur clusters and semiquinones in the NADH-Q oxidoreductase segment of the respiratory chain.

Our recent experimental data on iron-sulfur clusters and semiquinones in the complex I segment of the respiratory chain is presented, focusing on the Paracoccus (P.) denitrificans and bovine heart studies. The iron-sulfur cluster N2 has attracted the attention of investigators in the research field of complex I, since the mid-point redox potential of this cluster is the highest among all clusters in complex I, and is pH dependent (60 mV/pH). It is known that this cluster is located either in the NQO6 (NuoB in E. coli/PSST in bovine heart nomenclature) or in the NQO9 (NuoI/TYKY) subunit in the amphipathic domain of complex I. Our preliminary data indicate that the cluster N2 is located in the NuoB rather than the long-advocated NuoI subunit, and may have a unique ligand structure. We previously reported spin-spin interactions between cluster N2 and two distinct species of semiquinone (designated SQNf and SQNs) associated with complex I. A parallel intensity change was observed between the SQNf (g = 2.00) signal and the N2 split g parallel signal, further supporting our proposed interaction between SQNf and N2 spins.

Energy-dependent Complex I-associated ubisemiquinones in submitochondrial particles.

Two distinct species of Complex I-associated ubisemiquinones (SQNf and SQNs) were detected by cryogenic EPR analysis of tightly coupled submitochondrial particles oxidizing NADH or succinate under steady-state conditions. The g = 2.00 signals from both fast-relaxing SQNf (P1/2 = 170 mW at 40 K) and slow-relaxing SQNs (P1/2 = 0.7 mW) are sensitive to uncouplers, rotenone and thermally induced deactivation of Complex I. At higher temperatures the SQNf signal is broadened and only the SQNs signal is seen (P1/2 = 7 mW at 105 K). The spin-spin interaction between SQNf and the iron-sulfur cluster N2 was detected as split peaks of the g parallel 2.5 signal with a coupling constant of 1.65 mT, revealing their mutual distance of 8-11 A. The data obtained are consistent with a model in which N2 and two interacting bound ubisemiquinone species are spatially arranged within the hydrophobic domain of Complex I, participating in the vectorial proton translocation.

Coupling site I and the rotenone-sensitive ubisemiquinone in tightly coupled submitochondrial particles.

The rotenone-sensitive g = 2.00 low temperature EPR signal attributed to ubisemiquinone is observed in submitochondrial particles during coupled electron transfer from NADH to oxygen and from succinate to NAD+. The signal is seen only in the presence of oligomycin added to induce the respiratory control (7-9 with NADH and 3-4 with succinate) and it disappears in the presence of uncouplers (CCCP or gramicidin D). No reduction of the iron-sulfur center N-2 in the presence of 20 mM succinate and cyanide is observed, thus suggesting that N-2 is not in equilibrium with the ubiquinone pool. A hypothesis is proposed on delta mu H+ generation coupled with electron transfer between iron-sulfur center N-2 and the ubiquinone pool.

Studies on the conformational changes of metalloproteins induced by electrons in water-ethylene glycol solutions at low temperatures. III. Adrenal ferredoxin.

The reduction of adrenal ferredoxin (adrenodoxin) at low temperatures was investigated in order to separate local modifications of the active centre of the protein on its reduction, from the conformational transition which seems to accompany the change of the redox state of the irons; The ESR spectra of the states of the protein, where the reduced active centre is to be found by the "oxidized" conformation of the apoprotein, were obtained. The transition from the states of the protein to the state which occurs on its chemical reduction at room temperature was also investigated. The results of the work support the view that conformational changes in proteins (enzymes) which take place while they are functioning proceed after modifications of the active centres (change of the redox state, adsorption of a substrate, etc.), and are essentially caused by them. Adrenal ferredoxin was the third subject in our studies of the intermediate states of proteins which appear after reduction of their active centres by means of electrons trapped in water-ethylene glycol mixtures at the temperature of liquid nitrogen [1, 2]. In the reduced state, the active centre of the protein has an ESR signal with a g-factor of 1.94 [3, 4] which is convenient for our purposes.