Koyu Hon-Nami

Reversible thermal unfolding of thermostable cytochrome c-552

Abstract Reversible thermal denaturation of cytochrome c -552 from the extremely thermophilic bacterium Thermus thermophilus was studied by circular dichroism and fluorescence spectroscopy. Thermal denaturation in the presence of guanidine hydrochloride is completely reversible. The thermodynamic parameters for the reaction have been calculated based on a two-state mechanism. The free energy change on denaturation ( ΔG ) at 25 °C in the absence of denaturant is estimated to be 28.5 ± 0.15 kcal/mol, which is larger than that of cytochrome c from mesophilic organisms. The temperature of maximum stability is approximately 27 °C, which is higher than those of cytochromes c from mesophilic organisms (9 to 12 °C). The temperature dependences of the enthalpy and entropy changes are similar to those of cytochromes c from mesophilic organisms. The heat capacity change on denaturation is between 1250 and 1680 cal/deg mole, which is similar to those of cytochromes c from mesophilic organisms (1500 to 2500 cal/deg mol). From these results, it has been concluded that T. thermophilus cytochrome c is more stable than cytochromes c from mesophilic organisms by virtue of the fact that the free energy change for denaturation is greater and has its maximum at a higher temperature.

Cytochrome oxidase from an extreme thermophile, Thermusthermophilus HB 8

Abstract The cytochrome oxidase (EC 1.9.3.1) of Thermus thermophilus HB8 was isolated from the membrane fraction, and was highly purified. The oxidase contained heme a and heme c as the prosthetic groups. The purified preparation showed a single band in polyacrylamide gel electrophoresis, and three major polypeptides with apparent molecular weights of 52,000, 37,000 and 29,000 were observed in the presence of sodium dodecyl sulfate. The enzyme reacted rapidly with T . thermophilus cytochrome c-552. The oxidation of T . thermophilus cytochrome c -555,549 by the enzyme was very slow, and was stimulated by the addition of cytochrome c -552. The enzyme was highly stable to heat.

Purification, some properties and amino acid sequence of Thermus Thermophilus HB8 ferredoxin

A stable ferredoxin was purified in a crystalline form from an aerobic, thermophilic bacterium, Thermus thermophilus HB8. The molecular weight of the protein was determined to be 10500 by gel-filtration on Sephadex G-75 and to be 10200 by the sedimentation equilibrium method. The number of iron and acid labile sulfur atoms per mol was determined to be 6.3 and 6.4, respectively. The optical absorption spectrum of the ferredoxin has a broad maximum around 400 nm. The ferredoxin was so thermostable that its absorbance at 400 nm did not decrease after a 45-min incubation at 65 degrees C. The primary structure of the ferredoxin consisting of 78 amino acids was determined by sequence analysis of peptides obtained from a tryptic digest of the S-carboxymethylated ferredoxin and from a Staphylococcus aureus V8 protease digest of the S-aminoethylated derivative. The distribution of cysteine residues and the amino acid sequence around the cysteine residues are very similar to those of Mycobacterium smegmatis ferredoxin.

Macrocellulase complexes and yellow affinity substance from Clostridium thermocellum

Publisher Summary Clostridium thermocellum is an anaerobic thermophilic spore-forming bacterium. The preferred substrates for C. thermocellum are cellulose and cellobiose that are fermented to ethanol, acetate, lactate, CO 2 , and H 2 . The ability to ferment cellulose depends on an extracellular cellulolytic enzyme system, the formation of which is stimulated when the bacterium is grown on medium containing cellulose. The cellulolytic enzyme system consists of at least 14 different polypeptides that form complexes. C. thermocellurn during growth on cellulose synthesizes a yellow affinity substance (YAS) that attaches to the cellulose fibers to form YAS-cellulose. YAS seems to be intimately involved in the hydrolysis of cellulose. It facilitates the binding of the cellulolytic complexes to cellulose. This chapter describes methods for the isolation of the cellulolytic complexes and YAS as well as some of their properties.

Amino acid sequence of cytochrome c-552 from Thermus thermophilus HB8.

The complete amino acid sequence of thermophilic cytochrome c-552 from Thermus thermophilus HB8 is presented. The 131-residue sequence was derived by analysis of three cyanogen bromide fragments of the S-carboxymethylated apo-protein and their subpeptides. The sequence is homologous to c-type cytochromes, especially in the heme-binding region.

Cytochrome c-550 from a thermophilic bacterium PS3

Summary A c -type cytochrome, cytochrome c -550, was highly purified from a moderately thermophilic bacterium PS3. The absorption peaks were at 550, 521, and 416 nm in the reduced form, and at 408 nm in the oxidized form. The isoelectric point was at pH 5.6, and the molecular weight was about 10,000. The heat stability of the cytochrome was intermediate between those of horse cytochrome c and cytochrome c -552 from an extreme thermophile, Thermus thermophilus HB8.

Alkaline isomerization of thermoresistant cytochrome c-552 and horse heart cytochrome c studied by absorption and resonance Raman spectroscopy.

The structure of the thermoresistant cytochrome c (552, Thermus thermophilus) has been investigated at neutral and alkaline pH by absorption and resonance Raman spectroscopy and compared with that of horse heart cytochrome c. The ligands of the ferricytochrome c-552 at neutral pH are considered to be histidine and methionine, whereas the ligands of ferrocytochrome c-552 are histidine and another nitrogen base, histidine or lysine. Ferric cytochrome c-552 undergoes an alkaline isomerization with a pK of 12.3 (25 degrees C), accompanied by a ligand exchange. Horse heart cytochrome c has at least three isomerization states at alkaline pH (pK 9.3, 12.9 and greater than 13.5 at 25 degrees C). The replacement of the sixth ligand may not be involved in the second isomerization. The thermodynamic parameters for the isomerization were also estimated. The entropy change upon isomerization of cytochrome c-552 is negative, whereas for that of horse heart cytochrome c the entropy change is positive.

Kinetic studies on redox reactions of hemoproteins. I. Reduction of thermoresistant cytochrome c-552 and horse heart cytochrome c by ferrocyanide

The oxidation-reduction reaction of horse heart cytochrome c and cytochrome c (552, Thermus thermophilus), which is highly thermoresistant, was studied by temperature-jump method. Ferrohexacyanide was used as reductant. (Formula: see text.) Thermodynamic and activation parameters of the reaction obtained for both cytochromes were compared with each other. The results of this showed that (1) the redox potential of cytochrome c-552, + 0.19 V, is markedly less than that of horse heart cytochrome c. (2) deltaHox of cytochrome c-552 is considerably lower than that of horse heart cytochrome c. (3) deltaSox and deltaSred of cytochrome c-552 are more negative than those of horse heart cytochrome c. (4) kred of cytochrome c-552 is much lower than that of horse heart cytochrome c at room temperature.