A novel membrane-bound sulfide-oxidizing enzyme was purified 102-fold from the neutrophilic, obligately chemolithoautotrophic Thiobacillus sp. W5 by means of a six-step procedure. Spectral analysis revealed that the enzyme contains haem c and flavin. SDS-PAGE showed the presence of two types of subunit with molecular masses of 40 and 11 kDa. The smaller subunit contains covalently bound haem c, as was shown by haem staining. A combination of spectral analysis and the pyridine haemochrome test indicated that the sulfide-oxidizing heterodimer contains one molecule of haem c and one molecule of flavin. It appeared that the sulfide-oxidizing enzyme is a member of a small class of redox proteins, the flavocytochromes c, and is structurally most related to the flavocytochrome c sulfide dehydrogenase of the green sulfur bacterium Chlorobium limicola. The pH optimum of the enzyme is 8.6. At pH 9, the Vmax was 2.1 ± 0.1 μmol cytochrome c (mg protein)-1 min-1, and the Km values for sulfide and cytochrome c were 1.7 ± 0.4 μM and 3.8 ± 0.8 μM, respectively. Cyanide inhibited the enzyme by the formation of an N-5 adduct with the flavin moiety of the protein. On the basis of electron transfer stoichiometry, it seems likely that sulfur is the oxidation product.

Transient-state experiments with the obligately autotrophic Thiobacillus sp. strain W5 revealed that sulfide oxidation proceeds in two physiological phases, (i) the sulfate-producing phase and (ii) the sulfur-and sulfate-producing phase, after which sulfide toxicity occurs. Specific sulfur-producing characteristics were independent of the growth rate. Sulfur formation was shown to occur when the maximum oxidative capacity of the culture was approached. In order to be able to oxidize increasing amounts of sulfide, the organism has to convert part of the sulfide to sulfur (HS ؊ 3S 0 ؉ H ؉ ؉ 2e ؊) instead of sulfate (HS ؊ ؉ 4H 2 O3SO 4 2؊ ؉ 9 H ؉ ؉ 8e ؊), thereby keeping the electron flux constant. Measurements of the in vivo degree of reduction of the cytochrome pool as a function of increasing sulfide supply suggested a redox-related down-regulation of the sulfur oxidation rate. Comparison of the sulfur-producing properties of Thiobacillus sp. strain W5 and Thiobacillus neapolitanus showed that the former has twice the maximum specific sulfide-oxidizing capacity of the latter (3.6 versus 1.9 mol/mg of protein/min). Their maximum specific oxygen uptake rates were very similar. Significant mechanistic differences in sulfur production between the high-sulfur-producing Thiobacillus sp. strain W5 and the moderate-sulfur-producing species T. neapolitanus were not observed. The limited sulfide-oxidizing capacity of T. neapolitanus appears to be the reason that it can convert only 50% of the incoming sulfide to elemental sulfur.

Oligonucleotides, designed on the basis of conserved flanking amino acid sequence segments within the catalytic domain of eukaryotic protein kinase C (PKC) proteins, were used as primers for polymerase chain reactions to amplify a 427-bp chromosomal DNA fragment from the filamentous fungus Trichoderma reesei. This fragment was then used to isolate genes encoding PKC homologues of T. reesei and Aspergillus niger (pkc1 and pkcA, respectively). The genes contain six (T. reesei) and eight (A. niger) introns, which exhibit notable conservation in position with those found in the corresponding Schizosaccharomyces pombe pkc1+ and Drosophila melanogaster dPKC53Ebr genes. A single 4.2-kb transcript was detected in Northern analyses. The deduced PKC1 (T.reesei, 126 kDa) and PKCA (A. niger, 122 kDa) amino acid sequences reveal domains homologous to the C1 and C3/C4 domains of PKC-related proteins, but lack typical Ca(2+)-binding (C2) domains. Both contain a large, extended N-terminus, which shares a high degree of similarity with the corresponding regions of Saccharomyces cerevisiae PKC1 and S. pombe pkc1+ and pkc2+ proteins, but which is not present in PKCs of Dictyostelium or higher eukaryotes. This extended region can be divided into three subdomains; the N-terminal one contains a hydrophobic helix-turn-helix motif, whereas the C-terminal one contains potential targets for proteolytic processing. A polyclonal antiserum raised against the pseudosubstrate-binding domain of PKC1 recognizes in T. reesei a 115-120 kDa protein in Western blots. Expression of pkc1 cDNA in insect cells directs the synthesis of a PKC1 protein of similar size. The T. reesei PKC1 protein was partially purified and some of its properties examined: it is stimulated about twofold by phospholipids or phorbol esters but is not stimulated by Ca2+. We conclude that these PKC proteins from filamentous fungi represent the Ca(2+)-insensitive fungal homologues of the nPKC family.

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A highly active cytochrome c oxidase has been purified 75‐fold from the neutrophilic obligately autotrophic Thiobacillus sp. W5. UV/visible and electron paramagnetic resonance spectroscopy revealed that the cytochrome c oxidase contains low‐spin hemes c and low‐ and high‐spin hemes b. HPLC analysis confirmed the presence of heme b as the sole type of non‐covalently bound heme. The combined data from atomic absorption spectroscopy and electron paramagnetic resonance indicate the absence of CuA and suggest the presence of a bimetallic heme‐copper redox center. These results show that Thiobacillus sp. W5 contains a cbb3‐type oxidase, which is a member of the heme–copper oxidase family. The cbb3‐type oxidase was the only cytochrome oxidase expressed in aerobically and micro‐aerobically grown Thiobacillus sp. W5 cultures.