Electron Transfer and Catalytic Control by the Iron−Sulfur Clusters in a Respiratory Enzyme,<i>E.</i><i>coli</i>Fumarate Reductase

Hudson, Janette M.; Heffron, Kerensa; Kotlyar, Violetta; Sher, Yelizaveta; Maklashina, Elena; Cecchini, Gary; Armstrong, Fräser A.

doi:10.1021/ja043404q

Cited by 84 publications

(113 citation statements)

References 38 publications

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“…This pathway differs from the previously proposed bifurcated electron transfer pathway (58) for a greater number of thermodynamically unfavored steps. The latter are, however, possible within an overall thermodynamically favored reaction with precedents found in, for example, the well characterized fumarate reductase (59).…”

Section: Discussionmentioning

confidence: 88%

The Subnanometer Resolution Structure of the Glutamate Synthase 1.2-MDa Hexamer by Cryoelectron Microscopy and Its Oligomerization Behavior in Solution

Cottevieille

Larquet

Jonić

et al. 2008

Journal of Biological Chemistry

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The three-dimensional structure of the hexameric (␣␤) 6 1.2-MDa complex formed by glutamate synthase has been determined at subnanometric resolution by combining cryoelectron microscopy, small angle x-ray scattering, and molecular modeling, providing for the first time a molecular model of this complex ironsulfur flavoprotein. In the hexameric species, interprotomeric ␣-␣ and ␣-␤ contacts are mediated by the C-terminal domain of the ␣ subunit, which is based on a ␤ helical fold so far unique to glutamate synthases. The ␣␤ protomer extracted from the hexameric model is fully consistent with it being the minimal catalytically active form of the enzyme. The structure clarifies the electron transfer pathway from the FAD cofactor on the ␤ subunit, to the FMN on the ␣ subunit, through the low potential [4Fe-4S] 1؉/2؉ centers on the ␤ subunit and the [3Fe-4S] 0/1؉ cluster on the ␣ subunit. The (␣␤) 6 hexamer exhibits a concentration-dependent equilibrium with ␣␤ monomers and (␣␤) 2 dimers, in solution, the hexamer being destabilized by high ionic strength and, to a lower extent, by the reaction product NADP ؉ . Hexamerization seems to decrease the catalytic efficiency of the ␣␤ protomer only 3-fold by increasing the K m values measured for L-Gln and 2-OG. However, it cannot be ruled out that the (␣␤) 6 hexamer acts as a scaffold for the assembly of multienzymatic complexes of nitrogen metabolism or that it provides a means to regulate the activity of the enzyme through an as yet unknown ligand.Glutamate synthases (GltS) 2 are complex iron-sulfur flavoproteins that catalyze the reductive transfer of the L-Gln amide group to the C2 carbon of 2-oxoglutarate (2-OG), yielding two molecules of L-glutamate (L-Glu). GltS are found in bacteria, yeast, and plants, where they form with glutamine synthetase an essential pathway for ammonia assimilation (1-3). Bacterial NADPH-dependent GltS (NADPH-GltS) is formed by one ␣ subunit (␣GltS) and one ␤ subunit (␤GltS) of 162 and 52 kDa, respectively, for the Azospirillum brasilense GltS. The ␣␤ protomer contains one FAD (on ␤GltS), one FMN (on ␣GltS), and three different iron-sulfur clusters (one [3Fe-4S] 0/1ϩ cluster on ␣GltS and two [4Fe-4S] 1ϩ/2ϩ centers on ␤GltS; Fig. 1A). On the basis of sequence, structural, and mechanistic similarities, the NADPH-GltS serves as a model for the other two main forms of GltS, namely (i) the ferredoxin-dependent GltS found in cyanobacteria and photosynthetic tissues of plants, which is similar to ␣GltS and (ii) the eukaryotic type of GltS, which is NADH-dependent and is found in yeast, nonphotosynthetic tissues of plants, and lower eukaryotes; this GltS species is formed by a single polypeptide chain derived from the fusion of bacterial ␣ and ␤ subunits.Several lines of evidence support an essential role of GltS in all of these cell types, making it a potential target of novel drugs. For example, the NADPH-GltS has been found to be essential in Mycobacterium tuberculosis, where it has been shown that Ͻ1 M azaserine, a GltS inhibitor, is sufficient t...

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Section: Discussionmentioning

confidence: 88%

The Subnanometer Resolution Structure of the Glutamate Synthase 1.2-MDa Hexamer by Cryoelectron Microscopy and Its Oligomerization Behavior in Solution

Cottevieille

Larquet

Jonić

et al. 2008

Journal of Biological Chemistry

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“…S2C, suggests a cooperative, nonsimultaneous two-electron charge transfer process, i.e., one electron is transferred and then a second electron follows onto the same center (19)(20)(21)(22). We measure E m, 7 = −248 ± 1 mV, but using dcV it is very difficult to derive more precise mechanistic information regarding the separate one-electron processes that combine to give the "envelope" signal.…”

Section: Resultsmentioning

confidence: 99%

Electrochemical evidence that pyranopterin redox chemistry controls the catalysis of YedY, a mononuclear Mo enzyme

Adamson

Simonov

Kierzek

et al. 2015

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

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A long-standing contradiction in the field of mononuclear Mo enzyme research is that small-molecule chemistry on active-site mimic compounds predicts ligand participation in the electron transfer reactions, but biochemical measurements only suggest metal-centered catalytic electron transfer. With the simultaneous measurement of substrate turnover and reversible electron transfer that is provided by Fourier-transformed alternating-current voltammetry, we show that Escherichia coli YedY is a mononuclear Mo enzyme that reconciles this conflict. In YedY, addition of three protons and three electrons to the well-characterized "as-isolated" Mo(V) oxidation state is needed to initiate the catalytic reduction of either dimethyl sulfoxide or trimethylamine N-oxide. Based on comparison with earlier studies and our UV-vis redox titration data, we assign the reversible oneproton and one-electron reduction process centered around +174 mV vs. standard hydrogen electrode at pH 7 to a Mo(V)-to-Mo(IV) conversion but ascribe the two-proton and two-electron transition occurring at negative potential to the organic pyranopterin ligand system. We predict that a dihydro-to-tetrahydro transition is needed to generate the catalytically active state of the enzyme. This is a previously unidentified mechanism, suggested by the structural simplicity of YedY, a protein in which Mo is the only metal site.Fourier-transformed alternating-current voltammetry | mononuclear molybdenum enzyme | protein film electrochemistry | pyranopterin | YedY

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“…38 In the case of NiFe hydrogenase, the putative end of the relay system is the [4Fe4S] cluster that is coordinated by an unusual histidine ligand (H184 in Figure 29). Using sitedirected mutagenesis, H184 could be substituted for a cysteine.…”

Section: Interfacial and Intermolecular Electron Transfermentioning

confidence: 99%

“…The potential of this cluster is easily measured in noncatalytic experiments, as illustrated in Figure 26B, 38,40,66 where the outer trace is a voltammogram recorded at pH 8 in the absence of substrate. The faradaic current, obtained after subtracting the charging current, is shown in the same panel (not to scale).…”

Section: Intramolecular Electron Transfer (Iet)mentioning

confidence: 99%

Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies

2008

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Electron Transfer and Catalytic Control by the Iron−Sulfur Clusters in a Respiratory Enzyme,E.coliFumarate Reductase

Cited by 84 publications

References 38 publications

The Subnanometer Resolution Structure of the Glutamate Synthase 1.2-MDa Hexamer by Cryoelectron Microscopy and Its Oligomerization Behavior in Solution

The Subnanometer Resolution Structure of the Glutamate Synthase 1.2-MDa Hexamer by Cryoelectron Microscopy and Its Oligomerization Behavior in Solution

Electrochemical evidence that pyranopterin redox chemistry controls the catalysis of YedY, a mononuclear Mo enzyme

Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies

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