A method for preparing analytically pure sodium dithionite. Dithionite quality and observed nitrogenase-specific activities

McKenna, Charles E.; Gutheil, William G.; Song, Wei

doi:10.1016/0304-4165(91)90082-r

Cited by 64 publications

(58 citation statements)

References 32 publications

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“…Solutions of CYP3A4 in Nanodiscs were deaerated under the flow of argon gas and reduced by addition of a small excess of anaerobically prepared dithionite solution with the concentration determined using a molar absorption coefficient ⑀ 315 ϭ 8.05 mM Ϫ1 cm Ϫ1 (28). Complete reduction was confirmed by anaerobic absorption spectroscopy.…”

Section: Methodsmentioning

confidence: 99%

The Ferrous-Dioxygen Intermediate in Human Cytochrome P450 3A4

Denisov

Grinkova

Baas

et al. 2006

Journal of Biological Chemistry

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The oxy-ferrous complex is the first of three branching intermediates in the catalytic cycle of cytochrome P450, in which the total efficiency of substrate turnover is curtailed by the side reaction of autoxidation. For human membrane-bound cytochromes P450, the oxy complex is believed to be the primary source of cytotoxic superoxide and peroxide, although information on the properties and stability of this intermediate is lacking. Here we document stopped-flow spectroscopic studies of the formation and decay of the oxy-ferrous complex in the most abundant human cytochrome P450 (CYP3A4) as a function of temperature in the substrate-free and substrate-bound form. CYP3A4 solubilized in purified monomeric form in nanoscale POPC bilayers is functionally and kinetically homogeneous. In substrate-free CYP3A4, the oxy complex is extremely unstable with a half-life of ϳ30 ms at 5°C. Saturation with testosterone or bromocriptine stabilizes the oxy-ferrous intermediate. Comparison of the autoxidation rates with the available data on CYP3A4 turnover kinetics suggests that the oxy complex may be an important route for uncoupling.Cytochrome P450 CYP3A4 is the most prevalent P450 monooxygenase in human liver and is responsible for the metabolism of almost half of xenobiotics encountered by man (1). This isozyme has a large and flexible active site and is able to bind and catalytically convert multiple substrates, often displaying homotropic and heterotropic cooperativity in substrate binding and product formation. As a central player in human drug metabolism, CYP3A4 is one of the most intensely studied P450s, either in isolated human liver microsomes (2-5), detergent-solubilized form (6), or purified soluble aggregates (7,8). Recently, two groups have reported the crystal structure of a truncated CYP3A4 (9, 10). Despite this structural information, precise chemical and biophysical characterization of the human P450s has lagged that of the monomeric, soluble P450 isozymes isolated from bacteria. In particular, the critical intermediates in the reaction cycle of human P450s have not been precisely documented, due in part to the inability to form a robust, monomeric, and soluble entity that is amenable to the rapid reaction and spectroscopic methodologies successfully applied to the bacterial P450s. This lacuna is significant, as CYP3A4 and other human P450s display complex aspects of substrate recognition and catalytic mechanism that are not present in the simpler P450s such as CYP101 from Pseudomonas.The current version of the catalytic reaction cycle of cytochrome P450 monooxygenases involves intermediate reduction states of heme iron and atmospheric dioxygen and represents the result of over 30 years of intensive research (11,12). Traditionally, the cyclic process begins with the ferric low spin species and a water molecule occupying the sixth coordination site of the heme. In many cases the complementary fit of the native substrate into the pocket displaces this water ligand allowing the system to assume a predominantly hig...

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Section: Methodsmentioning

confidence: 99%

The Ferrous-Dioxygen Intermediate in Human Cytochrome P450 3A4

Denisov

Grinkova

Baas

et al. 2006

Journal of Biological Chemistry

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“…Sodium bicarbonate (NaHCO 3 ) was from Avantor Performance Materials. Sodium dithionite was purified to approximately 99% purity according to a published procedure (48). Gases were purchased from Air Liquide: CO 2 and acetylene.…”

Section: Methodsmentioning

confidence: 99%

Carbon dioxide reduction to methane and coupling with acetylene to form propylene catalyzed by remodeled nitrogenase

Yang

Moure

Dean

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

107

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A doubly substituted form of the nitrogenase MoFe protein (α-70 Val→Ala , α-195 His→Gln ) has the capacity to catalyze the reduction of carbon dioxide (CO 2 ) to yield methane (CH 4 ). Under optimized conditions, 1 nmol of the substituted MoFe protein catalyzes the formation of 21 nmol of CH 4 within 20 min. The catalytic rate depends on the partial pressure of CO 2 (or concentration of HCO 3 − ) and the electron flux through nitrogenase. The doubly substituted MoFe protein also has the capacity to catalyze the unprecedented formation of propylene (H 2 C = CH-CH 3 ) through the reductive coupling of CO 2 and acetylene (HC≡CH). In light of these observations, we suggest that an emerging understanding of the mechanistic features of nitrogenase could be relevant to the design of synthetic catalysts for CO 2 sequestration and formation of olefins.is an abundant and stable form of carbon that is the product of respiration and burning of fossil fuels. As a result of these activities, the atmospheric concentration of CO 2 , a greenhouse gas, has been rising over the last century and contributing to global warming (1). There is strong interest in developing methods for sequestering CO 2 either by capturing it or by chemically converting it to valuable chemicals (2-5). Of particular interest are possible routes to reduction of CO 2 by multiple electrons to yield methanol (CH 3 OH) and methane (CH 4 ), which are renewable fuels (2). The reduction of CO 2 is difficult, with a limited number of reports of metal-based compounds able to catalyze these reactions (6-13). In biology, only a few enzymes are known to reduce CO 2 (14-18), and none of these can catalyze the eight electron reduction to CH 4 .The bacterial Mo-dependent nitrogenase enzyme catalyzes the multielectron/proton reduction of dinitrogen (N 2 ) to two ammonia (NH 3 ) at a metal cluster designated FeMo-cofactor [7Fe-9S-1Mo-1C-R-homocitrate] (Fig. 1) in a reaction that requires ATP hydrolysis and evolution of H 2 , with a minimal reaction stoichiometry shown in Eq. 1 (19)(20)(21)(22).Given that nitrogenase is effective at catalyzing the difficult multielectron reduction of N 2 , it was of interest to determine whether this enzyme might also catalyze the reduction of CO 2 to the level of CH 4 . Nitrogenase is known to have the capacity to reduce a variety of other small, relatively inert, doubly or triply bonded compounds, such as acetylene (HC≡CH) (19,23). It has been shown that an alternative form of nitrogenase, which contains V in place of Mo in the active site cofactor, has the remarkable capacity to reduce CO and couple multiple CO molecules, yielding short chain alkenes and alkanes such as ethylene (C 2 H 4 ), ethane (C 2 H 6 ), propylene (C 3 H 6 ), and propane (C 3 H 8 ) (24,25). In contrast, the Mo-nitrogenase is only able to reduce CO at exceedingly low rates (24). However, we have found that the MoFe protein can be remodeled by substitution of amino acid residues that provide the first shell of noncovalent interactions with the active site FeMo cofa...

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“…Sodium dithionite was recrystallized to give Ͼ95% pure material according to the method described in the literature (12), adapted to be performed inside the anaerobic chamber. The stock solution of ferric-NTA was prepared as described in the literature at pH 7.0 (13).…”

Section: Methodsmentioning

confidence: 99%

Molecular Basis for Directional Electron Transfer

Paquete

Saraiva

Calçada

et al. 2010

Journal of Biological Chemistry

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Biological macromolecules involved in electron transfer reactions display chains of closely packed redox cofactors when long distances must be bridged. This is a consequence of the need to maintain a rate of transfer compatible with metabolic activity in the framework of the exponential decay of electron tunneling with distance. In this work intermolecular electron transfer was studied in kinetic experiments performed with the small tetraheme cytochrome from Shewanella oneidensis MR-1 and from Shewanella frigidimarina NCIMB400 using non-physiological redox partners. This choice allowed the effect of specific recognition and docking to be eliminated from the measured rates. The results were analyzed with a kinetic model that uses the extensive thermodynamic characterization of these proteins reported in the literature to discriminate the kinetic contribution of each heme to the overall rate of electron transfer. This analysis shows that, in this redox chain that spans 23 Å , the kinetic properties of the individual hemes establish a functional specificity for each redox center. This functional specificity combined with the thermodynamic properties of these soluble proteins ensures directional electron flow within the cytochrome even outside of the context of a functioning respiratory chain.Shewanella are facultative anaerobic ␥-proteobacteria capable of reducing a multitude of organic and inorganic substrates, including soluble and insoluble metallic compounds containing iron, manganese, uranium, or chromium (1). Shewanella arouse widespread interest in the science and in the engineering communities due to their role in geological phenomena such as global weathering and formation of minerals, their possible application in bioremediation of contaminated environments polluted with heavy metals, and their biotechnological applications for energy production in microbial fuel cells (1-3).The anaerobic respiratory flexibility found in these bacteria is associated with the presence of numerous multiheme cytochromes (4). One of the most abundant is the small tetraheme cytochrome (STC) 2 that has a 12-kDa molecular mass and contains four c-type hemes (5). Although the physiological role of STC in Shewanella oneidensis MR-1 (SoSTC) is still unknown, in S. frigidimarina NCIMB400 (SfSTC) it was shown to participate in iron respiration (6). The three-dimensional structure of SoSTC was determined for the reduced and oxidized states by x-ray crystallography (7), and recently the solution structure was solved for the reduced state of SfSTC (8). These studies showed that, despite the diversity found in the amino acid sequence of these cytochromes (64% identical), the architecture of their heme core is highly conserved with the hemes organized in a chain spanning 23 Å for the most distant heme irons (7,8). The determination of the structure of SoSTC led to the proposal that this protein may work as a nonspecific electron harvester (7). This proposal was refined by considering that hemes I-III would feed electrons to heme IV on t...

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A method for preparing analytically pure sodium dithionite. Dithionite quality and observed nitrogenase-specific activities

Cited by 64 publications

References 32 publications

The Ferrous-Dioxygen Intermediate in Human Cytochrome P450 3A4

The Ferrous-Dioxygen Intermediate in Human Cytochrome P450 3A4

Carbon dioxide reduction to methane and coupling with acetylene to form propylene catalyzed by remodeled nitrogenase

Molecular Basis for Directional Electron Transfer

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