Jorge Lampreia

Immunocytochemical localization of APS reductase and bisulfite reductase in three Desulfovibrio species

The localization of APS reductase and bisulfite reductase in Desulfovibrio gigas, D. vulgaris Hildenborough and D. thermophilus was studied by immunoelectron microscopy. Polyclonal antibodies were raised against the purified enzymes from each strain. Cells fixed with formaldehyde/glutaraldehyde were embedded and ultrathin sections were incubated with antibodies and subsequently labeled with protein A-gold. The bisulfite reductase in all three strains and APS reductase in d. gigas and D. vulgaris were found in the cytoplasm. The labeling of d. thermophilus with APS reductase antibodies resulted in a distribution of gold particles over the cytoplasmic membrane region. The localization of the two enzymes is discussed with respect to the mechanism and energetics of dissimilatory sulfate reduction.

ADENYLYLSULFATE REDUCTASES FROM SULFATE-REDUCING BACTERIA

Publisher Summary This chapter reviews adenylylsulfate (APS) reductases from sulfate-reducing bacteria (SRB). The sulfate molecule is inert chemically and must be activated in order to enter any of the pathways. All the organisms that utilize sulfate contain the enzyme ATP-sulfurylase, which catalyzes the formation of adenylylsulfate and inorganic pyrophosphate (PP i ) from ATP and sulfate. The APS molecule has twice the energy of the comparable ADP molecule, and the equilibrium of the reaction lies in the direction of ATP and sulfate. An inorganic pyrophosphatase shifts the equilibrium toward APS formation by hydrolyzing the PP i molecules. The key reaction in assimilatory sulfate reduction that differentiates it from dissimilatory sulfate reduction is the transferring of the sulfonate group of APS or 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to a thiol group producing the corresponding nucleotide (AMP or PAP) plus a thiosulfonate. This thiol is either a low molecular weight compound such as glutathione or a protein such as thioredoxin or glutaredoxin. The purification of APS reductase must be carried out in such a way that it maximizes its final purity and yet minimizes the time required to accomplish it.

Primary structure of desulfoferrodoxin from Desulfovibrio desulfuricans ATCC 27774, a new class of non-heme iron proteins

The primary structure of desulfoferrodoxin from Desulfovibrio desulfuricans ATCC 27774, a redox protein with two mononuclear iron sites, was determined by automatic Edman degradation and mass spectrometry of the composing peptides. It contains 125 amino acid residues of which five are cysteines. The first four, Cys‐9, Cys‐12, Cys‐28 and Cys‐29, are responsible for the binding of Center I which has a distorted tetrahedral sulfur coordination similar to that found in desulforedoxin from D. gigas. The remaining Cys‐115 is proposed to be involved in the coordination of Center II, which is probably octahedrally coordinated with predominantly nitrogen/oxygen containing ligands as previously suggested by Mössbauer and Raman spectroscopy.

Spectroscopic studies on APS reductase isolated from the hyperthermophilic sulfate-reducing archaebacterium Archaeglobus fulgidus

Adenylyl sulfate (APS) reductase, the key enzyme of the dissimilatory sulfate respiration, catalyzes the reduction of APS (the activated form of sulfate) to sulfite with release of AMP. A spectroscopic study was carried out with the APS reductase purified from the extremely thermophilic sulfate-reducing archaebacterium Archaeoglobus fulgidus DSM 4304. Combined ultraviolet/visible spectroscopy and low temperature electron paramagnetic resonance (EPR) studies were used in order to characterize the active centers and the reactivity towards AMP and sulfite of this enzyme. The A. fulgidus APS reductase is an iron-sulfur flavoprotein containing two distinct [4Fe-4S] clusters (Centers I and II) very similar to the homologous enzyme from Desulfovibrio gigas. Center I, which has a high redox potential, is reduced by AMP and sulfite, and Center II has a very negative redox potential.

Crystallization and preliminary X-ray analysis of a membrane-bound nitrite reductase from Desulfovibrio desulfuricans ATCC 27774

Nitrite reductase from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is a multihaem (type c) membrane-bound enzyme that catalyzes the dissimilatory conversion of nitrite to ammonia. Crystals of the oxidized form of this enzyme were obtained using PEG and CaCl(2) as precipitants in the presence of 3--(decylmethylammonium)propane-1-sulfonate and belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 78.94, b = 104.59, c = 143.18 A. A complete data set to 2.30 A resolution was collected using synchrotron radiation at the ESRF. However, the crystals may diffract to beyond 1.7 A and high-resolution data will be collected in the near future.

Preliminary crystallographic analysis and further characterization of a dodecaheme cytochrome c from Desulfovibrio desulfuricans ATCC 27774

Dodecaheme cytochrome c has been purified from Desulfovibrio (D.) desulfuricans ATCC 27774 cells grown under both nitrate and sulfate-respiring conditions. Therefore, it is likely to play a role in the electron-transfer system of both respiratory chains. Its molecular mass (37768 kDa) was determined by electrospray mass spectrometry. Its first 39 amino acids were sequenced and a motif was found between amino acids 32 and 37 that seems to exist in all the cytochromes of the c(3) type from sulfate-reducing bacteria sequenced at present. The midpoint redox potentials of this cytochrome were estimated to be -68, -120, -248 and -310 mV. Electron paramagnetic resonance spectroscopy of the oxidized cytochrome shows several low-spin components with a g(max) spreading from 3.254 to 2.983. Two crystalline forms were obtained by vapour diffusion from a solution containing 2% PEG 6000 and 0.25-0.75 M acetate buffer pH = 5.5. Both crystals belong to monoclinic space groups: one is P2(1), with a = 61.00, b = 106.19, c = 82.05 A, beta = 103.61 degrees, and the other is C2 with a = 152.17, b = 98.45, c = 89.24 A, beta = 119.18 degrees. Density measurements of the P2(1) crystals suggest that there are two independent molecules in the asymmetric unit. Self-rotation function calculations indicate, in both crystal forms, the presence of a non-crystallographic axis perpendicular to the crystallographic twofold axis. This result and the calculated values for the volume per unit molecular weight of the C2 crystals suggest the presence of two or four molecules in the asymmetric unit.