Extracellular laccases from submerged cultures of Coriolus versicolor BKM F-116, Panus tigrinus 8/18, Phlebia radiata 79 (ATCC 64658), Phlebia tremellosa 77-51 and from cultures of Pa. tigrinus 8/18, Ph. radiata 79 and Agaricus bisporus D-649 grown on wheat straw (solid-state fermentation) were purified. All enzymes from submerged cultures had a blue colour and characteristic absorption and EPR spectra. Laccases from the solid-state cultures were yellow-brown and had no typical blue oxidase spectra and also showed atypical EPR spectra. Comparison of N-terminal amino acid sequences of purified laccases showed high homology between blue and yellow-brown laccase forms. Formation of yellow laccases as a result of binding of lignin-derived molecules by enzyme protein is proposed.

The history of the elucidation of the microbiology and biochemistry of the oxidation of inorganic sulfur compounds in chemolithotrophic bacteria is briefly reviewed, and the contribution of Martinus Beijerinck to the study of sulfur-oxidizing bacteria highlighted. Recent developments in the biochemistry, enzymology and molecular biology of sulfur oxidation in obligately and facultatively lithotrophic bacteria are summarized, and the existence of at least two major pathways of thiosulfate (sulfur and sulfide) oxidation confirmed. These are identified as the 'Paracoccus sulfur oxidation' (or PSO) pathway and the 'S4intermediate' (or S4I) pathway respectively. The former occurs in organisms such as Paracoccus (Thiobacillus) versutus and P. denitrificans, and possibly in Thiobacillus novellus and Xanthobacter spp. The latter pathway is characteristic of the obligate chemolithotrophs (e.g. Thiobacillus tepidarius, T. neapolitanus, T. ferrooxidans, T. thiooxidans) and facultative species such as T. acidophilus and T. aquaesulis, all of which can produce or oxidize tetrathionate when grown on thiosulfate. The central problem, as yet incompletely resolved in all cases, is the enzymology of the conversion of sulfane-sulfur (as in the outer [S-] atom of thiosulfate [-S-SO3-]), or sulfur itself, to sulfate, and whether sulfite is involved as a free intermediate in this process in all, or only some, cases. The study of inorganic sulfur compound oxidation for energetic purposes in bacteria (i.e. chemolithotrophy and sulfur photolithotrophy) poses challenges for comparative biochemistry. It also provides evidence of convergent evolution among diverse bacterial groups to achieve the end of energy-yielding sulfur compound oxidation (to drive autotrophic growth on carbon dioxide) but using a variety of enzymological systems, which share some common features. Some new data are presented on the oxidation of 35S-thiosulfate, and on the effect of other anions (selenate, molybdate, tungstate, chromate, vanadate) on sulfur compound oxidation, including observations which relate to the roles of polythionates and elemental sulfur as intermediates.

Two different types of "Rieske" [2Fe-2S] clusters have been observed in proteins, one in the bc complexes of the respiratory chain and the other in bacterial dioxygenases. We have compared the circular dichroic (CD) spectra and redox properties of the water soluble fragment of the Rieske center of the bovine heart mitochondrial bc1 complex (ISF) and of the ferredoxin from benzene dioxygenase in Pseudomonas putida ML2 (FDBED). Spinach ferredoxin was also measured for comparison. The redox potential of both proteins could be determined in solution by cyclic voltammetry (CV) and by CD-monitored spectroelectrochemistry using a specially constructed optically transparent thin layer (OTTLE) cell. Whereas the redox potential of the ISF (+312 +/- 5 mV at pH 7.0) depended both on the pH above pH 7 and on the ionic strength, the redox potential of the FDBED (-155 +/- 5 mV at pH 7.0) was observed to be independent of pH and ionic strength. The ISF showed a marked dependence of its redox potential on temperature, while the FDBED showed no temperature dependence. The entropy of the redox reaction delta S degrees rc was calculated as -88 +/- 11 J K-1 mol-1 for the bc1 Rieske center and approximately 0 J K-1 mol-1 for the FdBED. The CD spectra of Rieske type clusters are significantly different from those of plant type [2Fe-2S] ferredoxins. A strong negative CD band is present at 20 000 cm-1 (500 nm) in all reduced Rieske clusters. The possible assignment of this band is discussed as arising from the highest energy magnetically allowed d --> d transition (dz2 --> dxz) of the FeII site. If so, this band is highly indicative of the distortion of the ligand field of the FeII site.

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