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PubMed1996

Spectroscopic states of the CO oxidation/CO2 reduction active site of carbon monoxide dehydrogenase and mechanistic implications.

CO dehydrogenases catalyze the reversible oxidation of CO to CO2, at an active site (called the C-cluster) composed of an Fe4S4 cube with what appears to be a 5-coordinate Fe (called FCII), linked to a Ni (Hu, Z., Spangler, N. J., Anderson, M. E., Xia, J., Ludden, P. W., Lindahl, P. A., & Münck, E. (1996) J. Am. Chem. Soc. 118, 830-845). During catalysis, electrons are transferred from the C-cluster to an [Fe4S4]2+/1+ electron-transfer cluster called the B-cluster. An S = 1/2 form of the C-cluster (called Cred1) converts to another S = 1/2 form (called Cred2) upon reduction with CO, at a rate well within the turnover frequency of the enzyme (Kumar, M., Lu, W.-P., Liu, L., & Ragsdale, S. W. (1993) J. Am. Chem. Soc. 115, 11646-11647). This suggests that the conversion is part of the catalytic mechanism. Dithionite is reported in this paper to effect this conversion as well, but at a much slower rate (kso = 5.3 x 10(-2) M-1 s-1 for dithionite vs 4.4 x 10(6) M-1 s-1 for CO). By contrast, dithionite reduces the oxidized B-cluster much faster, possibly within the turnover frequency of the enzyme. Dithionite apparently effects the Cred1/Cred2 conversion directly, rather than through an intermediate. The conversion rate varies with dithionite concentration. The Cred1/Cred2 conversion occurs at least 10(2) times faster in the presence of CO2 than in its absence. CO2 alters the g values of the gav = 1.82 signal, indicating that CO2 binds to a C-cluster-sensitive site at mild potentials. CN- inhibits CO oxidation by binding to FCII (Hu et al., 1996), and CO, CO2 in the presence of dithionite, or CS2 in dithionite accelerate CN- dissociation from this site (Anderson, M. E., & Lindahl, P. A. (1994) Biochemistry 33, 8702-8711). The effect of CO, CO2, and CS2 on CN- dissociation suggested that these molecules bind at a site (called the modulator) other than that to which CN- binds. The effects of CO2, CS2, CO, and dithionite on the Cred1/Cred2 conversion rate followed a similar pattern, suggesting that this rate is also influenced by modulator binding. Some batches of enzyme cannot convert to the Cred2 form using dithionite, but pretreatment with CO or CO2/dithionite effectively "cures" such batches of this disability. The results presented suggest that the Ni of the C-cluster is the modulator and the substrate binding site for CO/CO2. The inhibitor CS2 in the presence of dithionite also accelerates the decline of Cred1, leading first to an EPR-silent state of the C-cluster, and eventually to a state yielding an EPR signal with gav = 1.66. CS2 binding thus shares some resemblance to CO2 binding. Approximately 90% of the absorbance changes at 420 nm that occur when oxidized CODHCt is reduced by dithionite occur within 2 min at 10 degrees C. This absorbance change occurs in concert with the gav = 1.94 signal development. The remaining 10% of the A420 changes occur over the course of approximately 50 min, apparently coincident with the Cred1/Cred2 conversion. One possibility is that the conversion involves reduction of an (unidentified) Fe-S cluster. A three-state model of catalysis is proposed in which Cred1 binds and oxidizes CO, Cred2 is two electrons more reduced than Cred1 and is the state that binds and reduces CO2, and Cint is a one-electron-reduced state that is proposed to exist because of constraints imposed by the nature of the CO/CO2 reaction and the properties of the clusters involved in catalysis.

Aldehyde Oxidoreductases↗
PubMed2004

Removal of trichloroethylene in reduced soil columns.

A continuous soil column experiment was conducted to investigate reductive dechlorination of trichloroethylene (TCE) in soil system reductively manipulated by three types of reductants (Fe(II), dithionite, and Fe(II) + dithionite (combined treatment of Fe(II) and dithionite)). The soil column reduced by Fe(II) + dithionite has the greatest bed volumes (51.8) treated to breakthrough indicating that the combined treatment of Fe(II) and dithionite is more effective for the reductive dechlorination of TCE in the reduced soil column than the separate treatment of Fe(II) or dithionite. The measured bed volumes to breakthrough in control and treated soil columns were similar to the estimated bed volumes based on the result of batch kinetic experiment, differing by a factor of 0.96-1.02. The relative concentration of bromide (non-reactive tracer) reached the approximate value of 1 between 0.87 and 1.03 bed volume. C2 hydrocarbons (acetylene, ethylene, and ethane) were observed as transformation products in the effluents of soil columns treated by the reductants. However, no chlorinated intermediates were observed at the concentrations above detection limits throughout the experiment. Chloride was observed in the effluents of soil columns reduced by dithionite and Fe(II) + dithionite.

Chlorine↗
PubMed1989

Characterization of reductant-induced, tryptophan fluorescence changes in cytochrome oxidase.

We have measured the steady-state tryptophan fluorescence spectrum of cytochrome oxidase in its oxidized and fully reduced states. Reduction of the oxidized enzyme by sodium dithionite causes an apparent shift in the fluorescence emission maximum from 328 nm, in the oxidized enzyme, to 348 nm, in the reduced enzyme. This spectroscopic change has been observed previously and assigned to a redox-linked, conformational change in cytochrome oxidase [Copeland, R. A., Smith, P. A., & Chan, S. I. (1987) Biochemistry 26, 7311-7316]. When dithionite-reduced enzyme sits in an open cuvette, the enzyme returns to the oxidized state, and the fluorescence maximum shifts back to 328 nm. However, the time course of the fluorescence change does not follow the redox state of the enzyme, monitored spectrophotometrically at 445,605, and 820 nm, but follows the disappearance of dithionite, which absorbs at 315 nm. Moreover, when the fluorescence emission spectrum of the dithionite-reduced enzyme is corrected for the absorbance due to dithionite, the fluorescence maximum is found 2 nm blue shifted, relative to that of the oxidized enzyme, at 326 nm. This dithionite-induced, red-shifted steady-state tryptophan fluorescence is also seen with the non-heme-containing enzyme carboxypeptidase A. The tryptophan emission spectrum of untreated carboxypeptidase A is at 332 nm, whereas in the presence of dithionite the emission spectrum of carboxypeptidase A is at 350 nm. When corrected for the absorbance of dithionite, the tryptophan emission maximum is at 332 nm. We have also used the photoreductant 3,10-dimethyl-5-deazaisoalloxazine (deazaflavin) to reduce cytochrome oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Fluorescence assay for phospholipid membrane asymmetry.

Highly fluorescent 7-nitro-2,1,3-benzoxadiazol-4-yl-lipid (NBD-lipid) analogues are widely used to examine lipid transport and membrane structure. We have developed a method for chemically modifying NBD-labeled lipids in both artificial and biological membranes. This was achieved by treating fluorescently labeled membranes with dithionite (S2O4(-2)). When small unilamellar vesicles containing NBD-labeled phospholipids were reacted with dithionite, only the fluorescent lipid located on the outer leaflet of the vesicles' bilayer was reduced. Seven different NBD-lipid analogues, including a fluorescent sterol, were reduced by treatment with dithionite to nonfluorescent 7-amino-2,1,3-benzoxadiazol-4-yl-lipid derivatives. To assess the feasibility of using this reagent in biological systems, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dioleoylphosphatidylethanol ami ne was inserted into the outer leaflet of the plasma membrane of CHO-K1 cells. Subsequent incubation of these cells with a nontoxic concentration of dithionite resulted in the complete loss of fluorescence from the plasma membrane. In contrast, when cells were permitted to endocytose some of their fluorescently labeled plasma membrane and then treated with dithionite, fluorescence at the plasma membrane was eliminated, while intracellular labeling was not affected. These data suggest that dithionite reacts with NBD-labeled lipids in the outer leaflet of membrane bilayers, producing nonfluorescent derivatives. We demonstrate how reduction of NBD-lipids with dithionite can be used to prepare asymmetrically labeled liposomes and to measure transverse-membrane asymmetry in vesicles. This method should be useful in many biochemical investigations, including the measurement of phospholipid translocase activity.

4-Chloro-7-nitrobenzofurazan↗
PubMed1992

Transient-state kinetic evidence for intersubunit allosteric hysteresis during band 3 anion exchange.

On-line, dual-wavelength stopped-flow spectroscopy was used to follow continuously, the uptake of dithionite (S2O4(2-)) into human erythrocyte resealed ghosts. We describe the general characteristics of a pre-steady-state transient phase measured both with chloride and sulfate as co-anions. We then quantitatively characterize the dithionite concentration dependence of the amplitude factor and relaxation constant (kR) for the transient phase measured in sulfate medium. We also study the dithionite dependence of the steady-state velocity. Our results suggest that dithionite induces a slow conformational change in band 3 leading to hysteresis in the transport velocity. As many as 25 turnovers of the transport cycle per monomer can occur prior to attainment of steady state. Both kR and the amplitude factor for the transient phase were dithionite concentration dependent. In addition, the steady-state velocity showed apparent negative cooperativity. To discriminate between monomeric and intersubunit allosteric hysteresis, we performed a series of critical kinetic tests with cells labeled partially with 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). Coverage of 85% of the band 3 monomer population with DIDS caused kR to decrease about 10-fold, the dithionite concentration dependence of kR to change significantly, and the apparent negative cooperativity for the steady-state velocity to be eliminated. These results suggest that intersubunit allosteric hysteresis makes a significant contribution to dithionite transport by band 3.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1979

Studies on plant bile pigments, VII. Preparation and characterization of phycobiliproteins with chromophores chemically modified by reduction.

The reversible denaturation and reduction with dithionite has been studied for the phycobiliproteins, C-phycocyanin (1) and allophycocyanin (2) from Spirulina platensis, and C-phycoerythrin (4) from Fremyella diplosiphon (both cyanobacteria). By treatment with sodium dithionite, the chromophores are selectively reduced at the central (C-10) methine bridge, producing pigments with bilirubinoid (lambda max = 418 nm from 1 and 2), and vinylpyrroloc (lambda max= 300 nm from 4) chromophores. The extent of reduction is dependent on the state of the protein. The chromophores of denatured biliproteins are completely reduced at 0.5 mM dithionite. In the native pigments, dithionite concentrations up to 0.5 mM lead only to partial reduction, thus forming products containing both reduced and oxidized chromophores (e.g. "phycocyanorubins" from 1 and 2). The reduction is non-statistical with respect to the different chromophores present in 1 and 4, the chromophores absorbing at shorter wavelengths being preferentially reduced. Renaturation of the proteins containing reduced chromophores is accompanied by their reoxidation. This oxidation is complete in the absence of dithionite or at concentrations up to 0.5 mM. At higher dithionite concentrations, the reoxidation is incomplete, and the products are spectroscopically identical to those obtained by reduction of the native pigments at similar concentrations of reductant. The results are interpreted by a model in which the protein is "transparent" to the reducing agent, dithionite. The difference in the extent of reduction of the native and denatured pigments can only be due to thermodynamic (viz. stability) differences in the susceptibility of the chromophores to reduction. Specifically, the (extended) chromophore present in the native pigment is much more difficult to reduce than the chromophore (present in a cyclic conformation) in the denatured pigment. The energetics of the process of refolding both the protein and the chromophores are discussed.

Apoproteins↗
PubMed1994

Redox effects on the bacteriochlorophyll a-containing Fenna-Matthews-Olson protein from Chlorobium tepidum.

The BChl a-containing Fenna-Matthews-Olson (FMO) protein from the green sulfur bacterium Chlorobium tepidum was purified and characterized. Fluorescence spectra indicate that efficient excited state quenching occurs at neutral or oxidizing redox potentials. The major fluorescence lifetime at room temperature is approximately 60 ps in samples that are in neutral or oxidizing conditions, and approximately 2 ns in samples where the strong reductant sodium dithionite has been added. A similar change is observed in pump-probe picosecond absorbance difference experiments, where the long life time component increases after dithionite addition. A 16 Gauss wide EPR signal with g factor = 2.005 is observed in samples without dithionite. This signal largely disappears upon addition of dithionite. Dithionite induces large reversible changes in the 77 K absorbance spectra of the purified FMO protein and in whole cells. These results indicate that the FMO protein contains redox active groups, which may be involved in the regulation of energy transfer. Room temperature circular dichroism and low temperature absorption spectra show that dithionite also induces conformational or structural changes of the FMO protein complex.

Bacterial Proteins↗