Kinetic studies of the reduction of neutrophil cytochrome <i>b</i>-558 by dithionite

Aviram, Irit; Sharabani, Michaela

doi:10.1042/bj2370567

Cited by 7 publications

(5 citation statements)

References 27 publications

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“…The interpretation is clarified by another study that showed dithionite does not dissociate an analogous dimeric Grx–iron–sulfur complex under anaerobic conditions [39] .Therefore the most likely mechanism by which dithionite promotes dissociation of the dimeric Grx2 complex is by donating an electron to molecular oxygen to form superoxide which then acts as a one electron oxidant as shown in Scheme 1 . This conclusion is supported also by previous studies that documented formation of radicals (superoxide) when dithionite was added to aqueous solutions under aerobic conditions [65–67] .…”

Section: Discussionsupporting

confidence: 87%

Aging-dependent changes in rat heart mitochondrial glutaredoxins—Implications for redox regulation

et al. 2013

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Clinical and animal studies have documented that hearts of the elderly are more susceptible to ischemia/reperfusion damage compared to young adults. Recently we found that aging-dependent increase in susceptibility of cardiomyocytes to apoptosis was attributable to decrease in cytosolic glutaredoxin 1 (Grx1) and concomitant decrease in NF-κB-mediated expression of anti-apoptotic proteins. Besides primary localization in the cytosol, Grx1 also exists in the mitochondrial intermembrane space (IMS). In contrast, Grx2 is confined to the mitochondrial matrix. Here we report that Grx1 is decreased by 50–60% in the IMS, but Grx2 is increased by 1.4–2.6 fold in the matrix of heart mitochondria from elderly rats. Determination of in situ activities of the Grx isozymes from both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria revealed that Grx1 was fully active in the IMS. However, Grx2 was mostly in an inactive form in the matrix, consistent with reversible sequestration of the active-site cysteines of two Grx2 molecules in complex with an iron–sulfur cluster. Our quantitative evaluations of the active/inactive ratio for Grx2 suggest that levels of dimeric Grx2 complex with iron–sulfur clusters are increased in SSM and IFM in the hearts of elderly rats. We found that the inactive Grx2 can be fully reactivated by sodium dithionite or exogenous superoxide production mediated by xanthine oxidase. However, treatment with rotenone, which generates intramitochondrial superoxide through inhibition of mitochondrial respiratory chain Complex I, did not lead to Grx2 activation. These findings suggest that insufficient ROS accumulates in the vicinity of dimeric Grx2 to activate it in situ.

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Section: Discussionsupporting

confidence: 87%

Aging-dependent changes in rat heart mitochondrial glutaredoxins—Implications for redox regulation

et al. 2013

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“…The observed rate constant ( k obs ) did not increase linearly with the concentration of dithionite. Instead, as with other heme proteins, − the k obs showed a dependence on the square root of the dithionite concentration (Figure B) and plots of k obs versus [dithionite] 1/2 were linear (Figure B, inset). These results are consistent with the reductant being the sulfur dioxide radical anion (SO 2 •– ), formed by rapid homolysis of dithionite as described by eqs and . normalS 2 normalO 4 2 − ⇌ 2 SO 2 • − Fe(III)‐CBS + SO 2 • − → Fe(II)‐CBS + SO 2 The forward and reverse rate constants for eq are as follows: k 1 = 2.5 s –1 , and k –1 = 1.8 × 10 9 M –1 s –1 (25 °C). , This mechanism yields k obs as described by eq , where k 2 is the second-order rate constant for reduction of Fe(III)-CBS by SO 2 •– k obs = k 2 false( k 1 / k − 1 false) 1 / 2 false[ normalS 2 normalO 4 2 − false] 1 / 2 From the fit of the values of k obs to eq (Figure B), the dependence on [S 2 O 4 2– ] 1/2 was found to be k 2 ( k 1 / k –1 ) 1/2 = 353 ± 7 M –1/2 s –1 an...…”

Section: Resultsmentioning

confidence: 76%

Kinetics of Reversible Reductive Carbonylation of Heme in Human Cystathionine β-Synthase

Carballal

Cuevasanta²,

Marmisolle³

et al. 2013

Biochemistry

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Cystathionine β-synthase (CBS) catalyzes the condensation of homocysteine with serine or cysteine to form cystathionine and water or hydrogen sulfide (H2S), respectively. In addition to pyridoxal phosphate, human CBS has a heme cofactor with cysteine and histidine as ligands. While Fe(III)-CBS is inert to exogenous ligands, Fe(II)-CBS can be reversibly inhibited by carbon monoxide (CO) and reoxidized by O2 to yield superoxide radical. In this study, we have examined the kinetics of Fe(II)CO-CBS formation and reoxidation. Reduction of Fe(III)-CBS by dithionite showed square root dependence on concentration, indicating that the reductant species was the sulfur dioxide radical anion (SO2•−) that exists in rapid equilibrium with S2O42−. Formation of Fe(II)CO-CBS from Fe(II)-CBS and 1 mM CO occurred at (3.1 ± 0.4) × 10−3 s−1 (pH 7.4, 25 °C). The reaction of Fe(III)-CBS with the reduced form of the flavoprotein methionine synthase reductase in the presence of CO and NADPH resulted in its reduction and carbonylation to form Fe(II)CO-CBS. Fe(II)-CBS was formed as intermediate with a rate constant of (9.3 ± 2.5) × 102 M−1 s−1. Reoxidation of Fe(II)CO-CBS by O2 was multiphasic. The major phase showed hyperbolic dependence on O2 concentration. Although H2S is a product of the CBS reaction and a potential heme ligand, we did not find evidence for exogenous H2S effect on activity or heme binding. Reversible reduction of CBS by a physiologically relevant oxidoreductase is consistent with a regulatory role for the heme and could constitute a mechanism for crosstalk between the CO, H2S and superoxide signaling pathways.

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“…•¯ as the reducing agent (53)(54)(55)(56)(57)(58). Detailed analysis of the kinetics of reduction of P450 cam by dithionite was published by Hintz and Peterson (59), who found the mechanism of this reaction to involve SO 2 •¯ as the reducing species.…”

mentioning

confidence: 99%

“…Studies of the kinetics of reduction by dithionite were employed to probe apparent conformational transitions and changes in the ligand sphere of the heme iron in such heme-containing enzymes as bovine heart cytochrome c oxidase ( , ), cytochrome bo from Escherichia coli ( , ), and some other biological electron carriers ( − ). For most heme proteins studied it was shown that the mechanism of reduction involves the dithionite anion monomer SO •- 2 as the reducing agent ( − ). Detailed analysis of the kinetics of reduction of P450 cam by dithionite was published by Hintz and Peterson (), who found the mechanism of this reaction to involve SO •- 2 as the reducing species.…”

mentioning

confidence: 99%

Kinetics of Dithionite-Dependent Reduction of Cytochrome P450 3A4: Heterogeneity of the Enzyme Caused by Its Oligomerization

et al. 2005

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To explore the basis of apparent conformational heterogeneity of cytochrome P450 3A4 (CYP3A4), the kinetics of dithionite-dependent reduction was studied in solution, in proteoliposomes, and in Nanodiscs. In CYP3A4 oligomers in solution the kinetics obeys a three-exponential equation with similar amplitudes of each of the phases. Addition of substrate (bromocriptine) displaces the phase distribution toward the slow phase at the expense of the fast one, while the middle phase remains unaffected. The fraction reduced in the fast phase, either with or without substrate, is represented by the low-spin heme protein only, while the slow-reducible fraction is enriched in the high-spin CYP3A4. Upon monomerization by 0.15% Emulgen-913, or by incorporation into Nanodiscs or into large proteoliposomes with a high lipid-to-protein (L/P) ratio (726:1 mol/mol), the kinetics observed in the absence of substrate becomes very rapid and virtually monoexponential. In Nanodiscs and in lipid-rich liposomes bromocriptine decreases the rate of reduction via appearance of the second (slow) phase, the amplitude of which reaches 100% at saturating bromocriptine. In contrast, in P450-rich liposomes (L/P = 112 mol/mol), where the surface molar density of the enzyme is comparable to that observed in liver microsomes, CYP3A4 behaves similarly to that observed in solution. These results suggest that in CYP3A4 oligomers in solution and in the membrane the enzyme is distributed between two persistent conformers with different accessibility of the heme for the reductant (SO*-(2) anion monomer). One of the apparent conformers exists in a substrate-dependent equilibrium between two states with different rate constants of reduction by dithionite, while the second conformer shows no response to substrate binding.

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Kinetic studies of the reduction of neutrophil cytochrome b-558 by dithionite

Cited by 7 publications

References 27 publications

Aging-dependent changes in rat heart mitochondrial glutaredoxins—Implications for redox regulation

Aging-dependent changes in rat heart mitochondrial glutaredoxins—Implications for redox regulation

Kinetics of Reversible Reductive Carbonylation of Heme in Human Cystathionine β-Synthase

Kinetics of Dithionite-Dependent Reduction of Cytochrome P450 3A4: Heterogeneity of the Enzyme Caused by Its Oligomerization

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