(Kcccived October 25, 1991) -EJB 91 1438In addition to the 50-kDa (a) and 40-kDa (0) subunits, a n 11-kDa polypeptide has been discovered in highly purified Desulfovihrio vulguris (Hildenborough) dissimilatory sulfite reductase. This is in contrast with the hitherto generally accepted u2p2 tetrameric subunit composition. Purification, high-ionic-strength gel-filtration, native electrophoresis and isoelectric focussing d o not result in dissociation of the 11-kDa polypeptide from the complex. Densitometric scanning of SDS gels and denaturing gel-filtration indicate a stoichiometric occurrence. A similar 1 I-kDa polypeptide is present in the desulfoviridin of D. vulgaris oxarnicus (Monticello), D . gigas and D. dcsulfuricuns ATCC 27774. We attribute an cc,D,y, subunit structure to desulfoviridin-type sulfite reductases. N-terminal sequences of the a, / 3 and y subunits are reported.A key enzyme in dissimilatory sulfate reduction is sulfite reductase, a complex redox enzyme containing both Fe/S and siroheme prosthetic groups. Sulfite reductases that are supposed to have a dissimilatory function have been observed in, and isolated from, over 20 species of microorganisms [ l -41. Kinetic parameters and subunit structure of dissimilatory sulfite reductases are markedly different from assimilatory sulfite reductases.Dissimilatory enzymes are x 2 P 2 proteins (1 50-230 kDa) that have a millimolar K,,, for sulfite, a slightly acidic pH optimum and reduce their substrate to SJOzSp and S20:-(1, 4-81, The physiological relevance of the in vitro observed product composition is a matter of controversy. A number of explanations has been put forward: loss of interaction with the cytoplasmic membrane [9], in vitro assay conditions [lo], and the possibility that the observed products are in vivo intermediates [I 11. Partial demctallation of the siroheme is observed in desulfoviridins [2,12] and is presumably an intrinsic feature of some dissimilatory enzymes. It probably has no bearing on the formation of S 3 0 i -and SzO:-since desulfoviridin, desulfofuscidin, P582 and desulforubidin-type dissimilatory enzymes have a comparable product composition and specific activity [2-7, 131.Contrarily, assimilatory sulfite reductases cleanly perform the full six-electron reduction to sulfide. They have a slightly basic pH optimum with a submillimolar K,, for sulfite. The subunit composition, however, is non-uniform: ugp4 [14], a4-6 1351, or cc2 [16]. This probably reflects differences in the source of reducing equivalents.A distinct, third, group of sulfitc reductases comprises the so-called low-molecular-mass sulfite reductases. These enzymes arc presumably assimilatory. They differ from the other two groups in two aspects: they are monomeric (20 -30 kDa), We report here that D . vulgaris (Hildenborough) desulfoviridin contains a small, hitherto unnoted, y-subunit in addition to the usual a2P2 motive of dissimilatory enzymes. Nterminal sequences, size and stoichiometry of the subunits have been determined. An immunological comparison of d...
Nitrogenase consists of two metalloproteins (Fe protein and MoFe protein) which are assumed to associate and dissociate to transfer a single electron to the substrates. This cycle, called the Fe protein cycle, is driven by MgATP hydrolysis and is repeated until the substrates are completely reduced. The rate-limiting step of the cycle, and substrate reduction, is suggested to be the dissociation of the Fe protein-MoFe protein complex which is obligatory for the reduction of the Fe protein [Thorneley, R. N. F., and Lowe, D. J. (1983) Biochem. J. 215, 393-403]. This hypothesis is based on experiments with dithionite as the reductant. We also tested besides dithionite flavodoxin hydroquinone, a physiological reductant. Two models could describe the experimental data of the reduction by dithionite. The first model, with no reduction of Fe protein bound to MoFe protein, predicts a rate of dissociation of the protein complex of 8.1 s-1. This rate is too high to be the rate-limiting step of the Fe protein cycle (kobs = 3.0 s-1). The second model, with reduction of the Fe protein in the nitrogenase complex, predicts a rate of dissociation of the protein complex of 2.3 s-1, which in combination with reduction of the nitrogenase complex can account for the observed turnover rate of the Fe protein cycle. When flavodoxin hydroquinone (155 microM) was the reductant, the rate of reduction of oxidized Fe protein in the nitrogenase complex (kobs approximately 400 s-1) was 100 times faster than the turnover rate of the cycle with flavodoxin as the reductant (4 s-1). Pre-steady-state electron uptake experiments from flavodoxin hydroquinone indicate that before and after reduction of the nitrogenase complex relative slow reactions take place, which limits the rate of the Fe protein cycle. These results are discussed in the context of the kinetic models of the Fe protein cycle of nitrogenase.
MgATP-dependent pre-steady-state proton production by nitrogenase from Azotobacter vinelandii was studied by monitoring the absorbance changes at 572 nm of the pH indicator o-cresolsulphonphtalein in a weakly buffered solution. The absorbance changes are characterized by a constant phase, a single exponential decrease and a linear decrease. The observed rate constant for the single exponential MgATP-dependent proton production by reduced nitrogenase proteins at 20.0 "C is 14 2 4 s-'. No proton production with a rate constant comparable to the observed rate constant of electron transfer (/coba = 100 s-l) was detected. The extent of the observed MgATP-dependent proton production is determined by the redox state of the nitrogenase proteins before mixing with MgATP; less protons are produced when more electrons are transferred from the Fe protein to the MoFe protein. Values of 2.7 +-0.3 mol H&duced/mOl MoFe protein with oxidized Fe protein, and 1.1 2 0.1 mol H&duced/mol MoFe protein with reduced Fe protein, were found. The data are interpreted to mean that protons are taken up after electron transfer from the Fe protein to the MoFe protein; the ratio electronsuansre,,ed~/H,+,,,, was calculated to be 1.2 2 0.2.After mixing the nitrogenase proteins with MgADP, proton production takes place as well. The proton-production curve did not have a constant phase and the observed rate constant of the single exponential reaction is higher, compared to MgATP-dependent proton production (koba = 35 sP).The amount of protons produced depends also on the redox state of the Fe protein; no proton production was observed with the oxidized Fe protein ; with dithionite-reduced Fe protein a value of 3.1 2 0.4 mol H&,duCed/mol MoFe protein was found (or 0.5 2 0.1 mol H+/mol Fe protein). Similar results were obtained when only the Fe protein was mixed with MgADP, but the observed absorbance changes were smaller; mixing of dithionite-reduced Fe protein with MgADP resulted in the production of 0.17 2 0.05 mol H+/mol Fe protein.All reported absorbance changes were absent when the experiments were performed in a buffered solution.The series of events that occur after mixing of the nitrogenase proteins with MgATP will be presented and discussed. In the case of the reduced Fe protein, electron transfer takes place at a rate of 100 s-', which is followed by H' production (kobs = 14 ss'). When there is no electron transfer (oxidized Fe protein) the rate constant of the MgATP-induced proton production decreases. When electrons are transferred, stoichiometrically less protons are produced.Nitrogenase is the enzyme system which catalyses the reaction of nitrogen fixation, in which dinitrogen is reduced to ammonia. Nitrogenase consists of two distinct oxygensensitive metalloproteins, which are both necessary for catalytic activity [l -31. The molybdenum-containing nitrogenase was studied in the present investigation. The largest of the nitrogenase proteins is the MoFe protein (Avl); it contains two types of metal-sulphur clusters, an Fe-, Mo-and Scon...
A method for the quantitative determination of riboflavin levels in beer was developed. The method is based on the quenching of riboflavin fluorescence, which occurs when riboflavin binds to the aporiboflavin-binding protein from egg white. The method does not require any pretreatment of the beer before analysis, other than dilution, and proved to be simple, reliable, and sensitive. The lowest concentration that could be detected was approximately 10 nM riboflavin. The possible interference of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) with the determination of the riboflavin content of beer was excluded, because beer contains only a very small amount of FAD (0.03 microM) and no FMN. The riboflavin levels of the types and brands of beer investigated were in the range of 0.5-1.0 microM. The origin of the riboflavin in beer proved to be the malt. Hop and yeast hardly contributed to the riboflavin content of beer. Besides its use in the determination of riboflavin levels, the aporiboflavin-binding protein also provides a way to remove riboflavin from beer, which reduces the light sensitivity and the related lightstruck off-flavor formation in beer.
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