Appl. Environ. Microbiol. 63:896-902, 1997). This solute was purified after extraction from the cell biomass. In addition, the optically active and the optically inactive (racemic) forms of the compound were synthesized, and the ability of the solute to act as a protecting agent against heating was tested on several proteins derived from mesophilic or hyperthermophilic sources. Diglycerol phosphate exerted a considerable stabilizing effect against heat inactivation of rabbit muscle lactate dehydrogenase, baker's yeast alcohol dehydrogenase, and Thermococcus litoralis glutamate dehydrogenase. Highly homologous and structurally well-characterized rubredoxins from Desulfovibrio gigas, Desulfovibrio desulfuricans (ATCC 27774), and Clostridium pasteurianum were also examined for their thermal stabilities in the presence or absence of diglycerol phosphate, glycerol, and inorganic phosphate. These proteins showed different intrinsic thermostabilities, with half-lives in the range of 30 to 100 min. Diglycerol phosphate exerted a strong protecting effect, with approximately a fourfold increase in the half-lives for the loss of the visible spectra of D. gigas and C. pasteurianum rubredoxins. In contrast, the stability of D. desulfuricans rubredoxin was not affected. These different behaviors are discussed in the light of the known structural features of rubredoxins. The data show that diglycerol phosphate is a potentially useful protein stabilizer in biotechnological applications.One of the most striking characteristics of extremophiles is their ability to thrive under environmental conditions that would be lethal to most organisms. In particular, hyperthermophiles, having optimal growth temperatures above 80°C (4), pose intriguing questions regarding the biochemical strategies used for the adaptation of their cellular structures to withstand such high temperatures. Maintaining protein performance at high temperature could be accomplished by a number of mechanisms: (i) intrinsic thermostability, (ii) increased turnover, (iii) improved action of molecular chaperones, and (iv) extrinsic stabilization by compatible solutes (13). Although most enzymes from thermophilic sources show an intrinsic thermostability higher than that of their mesophilic counterparts, several enzymes derived from hyperthermophilic sources show an unexpectedly low intrinsic stability in vitro (13,14). Therefore, extrinsic factors, such as compatible solutes, may play a role in the thermostabilization of these cellular components.Some compatible solutes, namely, glutamate, betaine, and trehalose, are widespread in mesophilic organisms, but compatible solutes unique to thermophiles and hyperthermophiles have also been identified in recent years (8; H. Santos and M. S. da Costa, submitted for publication). Newly discovered solutes from thermophilic and hyperthermophilic organisms include cyclic-2,3-bisphosphoglycerate (17), two isomers of dimyo-inositol phosphate (25, 31), mannosylglycerate and mannosylglyceramide (24, 28, 36), di-mannosyl-di-myo-inosito...
Two new multiheme cytochromes were isolated from the anaerobic sulfur reducing bacterium Desulfuromonas acetoxidans. They have monomeric molecular masses of 50 and 65 kDa and contain six and eight hemes, respectively. Visible and EPR spectroscopies, in the as-isolated (oxidised) cytochromes, show the presence of only low-spin hemes in the 50-kDa cytochrome, and of high-spin and low-spin hemes in the 65-kDa cytochrome. The EPR spectra of the native 65-kDa cytochrome indicate multiple heme-heme interactions, including integer-spin systems as judged by parallel-mode EPR. The 50-kDa cytochrome has a complex redox pattern, as shown by EPR redox titrations, and contains one heme with unusual characteristics. Both cytochromes cover an extremely wide range of reduction potentials, which go from +100 mV to -375 mV for the 50-kDa cytochrome, and +I85 mV to -235 mV for the 65-kDa cytochrorne.The two cytochromes were tested for hydroxylamine oxidoreductase activity and polysulfide reductase activity, but neither displayed any activity. In contrast, it was found for the first time that the previously characterised cytochrome c,,, ,, from the same bacterium is very active in the reduction of polysulfide, which suggests that it acts as a terminal reductase in D. acetoxidans.
A c-type monoheme ferricytochrome csso (9.6 kDa) was isolated from cells of Bacillus halodenitrzjkans sp.nov., grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme-methionine coordination. The midpoint redox potential was determined at several pH values by visible spectroscopy. The redox potential at pH 7.6 is 138 mV.When studied by 'H-NMR spectroscopy as a function ofpH, the spectrum shows a pH dependence with pK, values of 6.0 and 11 .O. According to these pK, values, three forms designated as I, I1 and I11 can be attributed to cytochrome ~5 5 0 . The first pK, is probably associated with protonation of the propionate groups. The second pK, value introduces a larger effect in the 'H-NMR spectrum and is probably due to the ionisation of the axial histidine. Studies of temperature variation of the 'H-NMR spectra for both the ferrous and ferri forms of the cytochrome were performed. Heme meso protons, the heme methyl groups, the thioether protons, two protons from a propionate and the inethylene protons from the axial methionine were identified in the reduced form. The heme methyl resonances of the ferri form were also assigned. EPR spectroscopy was also used to probe the ferric heme environment. A signal at g,,, z 3.5 at pH 7.5 was observed indicating an almost axial heme environment. At higher pH values the signal at g,,, = 3.5 converts mainly to a signal at g z 2.96. The pK, associated with this change is around 11.3.The N-terminal sequence of this cytochrome was determined and compared with known amino acid sequences of other cytochromes.Bacillus halodenitricans sp. nov. (ATCC 49067) tolerates high concentrations of nitrate, nitrite and sodium ions and produces nitrous oxide rather than dinitrogen as the end product of nitrate reduction [I]. The nitrate reductase and nitrite reductase from this organism were purified and characterised (2, 31. The nitrate reductase is a membrane bound, hemecontaining, molybdo-iron protein that oxidises menadiol and reduces nitrate to nitrite. The nitrite reductase is a coppercontaining enzyme strongly bound to the cytoplasmic membrane.The electron transfer components of the denitrification pathway include cytochrome b559, a cytochrome c550 and a copper nitrite reductase [3,4]. Both cytochromes are involved in the reduction of nitrate to nitrous oxide, but only cytochrome b5s9 lies in the pathway of nitrate reduction to nitrite.Cytochrome c5s0 was shown to be involved in the reduction of nitrite by membrane vesicles [4]. It can not replace phenazine methosulfate as electron mediator between ascorbate and purified nitrite reductase in activity assays [4], and ascorbate-reduced cytochrome ~5 5 0 is not reoxidised in Correspondcnce to
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