Ming-Y. Liu

Thermostabilization of Proteins by Diglycerol Phosphate, a New Compatible Solute from the Hyperthermophile Archaeoglobus fulgidus

ABSTRACT Diglycerol phosphate accumulates under salt stress in the archaeonArchaeoglobus fulgidus (L. O. Martins, R. Huber, H. Huber, K. O. Stetter, M. S. da Costa, and H. Santos, 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, bakers yeast alcohol dehydrogenase, andThermococcus litoralis glutamate dehydrogenase. Highly homologous and structurally well-characterized rubredoxins fromDesulfovibrio 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. pasteurianumrubredoxins. In contrast, the stability of D. desulfuricansrubredoxin 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.

Regulation of the hexaheme nitrite/nitric oxide reductase of Desulfovibrio desulfuricans, Wolinella succinogenes and Escherichia coli: A mass spectrometric study

Dissimilatory nitrite reduction, carried out by hexaheme proteins, gives ammonia as the final product. Representatives of this enzyme group from 3 bacterial species can also reduce NO to either ammonia or N2O. The redox regulation of the nitrite/nitric oxide activities is discussed in the context of the denitrifying pathway.

Isolation and Preliminary Characterization of a Soluble Nitrate Reductase from the Sulfate Reducing Organism Desulfovibrio desulfuricans ATCC 27774

Desulfovibrio desulfuricans ATCC 27774 is a sulfate reducer that can adapt to nitrate respiration, inducing the enzymes required to utilize this alternative metabolic pathway. Nitrite reductase from this organism has been previously isolated and characterized, but no information was available on the enzyme involved in the reduction of nitrate. This is the first report of purification to homogeneity of a nitrate reductase from a sulfate reducing organism, thus completing the enzymatic system required to convert nitrate (through nitrite) to ammonia. D. desulfuricans nitrate reductase is a monomeric (circa 70 kDa) periplasmic enzyme with a specific activity of 5.4 K(m) for nitrate was estimated to be 20 microM. EPR signals due to one [4Fe-4S] cluster and Mo(V) were identified in dithionite reduced samples and in the presence of nitrate.

Localization and specificity of cytochromes and other electron transfer proteins from sulfate-reducing bacteria

Recently data have accumulated concerning the electron transfer chains of sulfate-reducing bacteria in general and of the genus Desulfovibrio in particular. Because of the ever growing number of newly discovered individual redox proteins, it has become essential to try to assign them to physiologically relevant chains. This work presents some new data concerning the localization of these proteins within the bacterial cell and the specificity of electron transfer between the three types of hydrogenases which have been found so far in Desulfovibrio, namely the iron-only, the iron-nickel and the iron-nickel-selenium enzymes. The iron-only hydrogenase reduces cytochromes which have bis-histidinyl heme ligation or histidinyl-methionyl heme ligation. In contrast, the iron-nickel and iron-nickel-selenium hydrogenases cannot reduce cytochromes having a His-Met heme ligation, but are very active toward the cytochromes having a bis-histidinyl ligand. This observation has been used to demonstrate that the tetraheme cytochrome c3 can exchange electrons with the monoheme cytochrome c553. No clear specificity has been established for the reaction of hydrogenases toward the hexadecaheme cytochromes from either D vulgaris or D gigas.

Structural and functional approach toward a classification of the complex cytochrome c system found in sulfate-reducing bacteria

Following the discovery of the tetraheme cytochrome c3 in the strict anaerobic sulfate-reducing bacteria (Postgate, J.R. (1954) Biochem. J. 59, xi; Ishimoto et al. (1954) Bull. Chem. Soc. Japan 27, 564-565), a variety of c-type cytochromes (and others) have been reported, indicating that the array of heme proteins in these bacteria is complex. We are proposing here a tentative classification of sulfate- (and sulfur-) reducing bacteria cytochromes c based on: number of hemes per monomer, heme axial ligation, heme spin state and primary structures (whole or fragmentary). Different and complementary spectroscopic tools have been used to reveal the structural features of the heme sites.

Purification and characterization of two proteins with inorganic pyrophosphatase activity from Desulfovibriovulgaris: Rubrerythrin and a new, highly active, enzyme

The inorganic pyrophosphatase activity of a soluble extract from the strict anaerobe, sulfate-reducing, Desulfovibrio vulgaris, is readily resolved into two peaks. After purification, two active proteins with very dissimilar properties are obtained. One is the non-heme iron-containing rubrerythrin, with a specific activity of 350 pyrophosphate hydrolyzed, min-1, mg protein-1. The other, a protein of Mr = 39,000, with a specific activity of 12,000.

Comparative EPR studies on the nitrite reductases from Escherichia coli and Wolinella succinogenes

Hexaheme nitrite reductases purified to homogeneity from Escherichia coli K‐12 and Wolinella succinogenes were studied by low‐temperature EPR spectroscopy. In their isolated states, the two enzymes revealed nearly identical EPR spectra when measured at 12 K. Both high‐spin and low‐spin ferric heme EPR resonances with g values of 9.7, 3.7, 2.9, 2.3 and 1.5 were observed. These signals disappeared upon reduction by dithionite. Reaction of reduced enzyme with nitrite resulted in the formation of ferrous heme‐NO complexes with distinct EPR spectral characteristics. The heme‐NO complexes formed with the two enzymes differed, however, in g values and line‐shapes. When reacted with hydroxylamine, reduced enzymes also showed the formation of ferrous heme‐NO complexes. These results suggested the involvement of an enzyme‐bound NO intermediate during the six‐electron reduction of nitrite to ammonia catalyzed by these two hexaheme nitrite reductases. Heme proteins that can either expose bound NO to reduction or release it are significant components of both assimilatory and dissimilatory metabolisms of nitrate. The different ferrous heme‐NO complexes detected for the two enzymes indicated, nevertheless, their subtle variation in heme reactivity during the reduction reaction.

Calcium is required for the reduction of sulfite from hydrogen in a reconstituted electron transfer chain from the sulfate reducing bacterium, Desulfovibriogigas

Calcium is found a strong stimulator of sulfite reduction from hydrogen. A coupling protein of molecular weight 65,000 can be isolated from Desulfovibrio gigas. It functions in a reconstituted electron transfer chain between hydrogenase and sulfite reductase. Its N-terminal sequence shows high homologies with calcium or magnesium binding sites from other calcium-binding proteins.

Characterization of Electron Transfer Proteins

The end-product of their respiration, hydrogen sulfide, is making sulfate-reducing bacteria (SRB) one of the main microbial agent that interacts with metals through formation of sulfide. As a consequence, most of the metals which are found to interact with living organisms have been found in these bacteria, with the exception of copper metal probably because the redox potentials of the Cu+/Cu2+ complexes are too high to be compatible with any of the physiological reactions common to SRB.

Probing the iron environment in desulforedoxin. EXAFS of oxidized and reduced states

Abstract Fe XAS data were collected on the oxidized and reduced forms of desulforedoxin from Desulfovibrio gigas, the oxidized form of rubredoxin from Clostridium pasteurianum, and the reduced form of rubredoxin from Pyrococcus furiosus. Analysis of these data is consistent with tetrahedral FeS4 coordination in both oxidation states, and an expansion of the Fe-S distances from 2.27 to 2.33 A upon reduction.