Synthetic Analogues of the Active Sites of Iron−Sulfur Proteins

Rao, P. Venkateswara; Holm, R. H.

doi:10.1021/cr020615+

Cited by 520 publications

(352 citation statements)

References 343 publications

(639 reference statements)

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“…We argue, as has been previously proposed (10,11), that the catalytically active form contains one [4Fe-4S] 2ϩ cluster and that the other cluster species are artifacts of the reconstitution (33). Spectroscopically, such species are not expected to be discernible from genuine Fe/S cofactors.…”

Section: Discussionsupporting

confidence: 61%

Escherichia coli L-Serine Deaminase Requires a [4Fe-4S] Cluster in Catalysis

Cicchillo¹,

Baker²,

Schnitzer³

et al. 2004

Journal of Biological Chemistry

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L-Serine deaminases catalyze the deamination of Lserine, producing pyruvate and ammonia. Two families of these proteins have been described and are delineated by the cofactor that each employs in catalysis. These are the pyridoxal 5 -phosphate-dependent deaminases and the deaminases that are activated in vitro by iron and dithiothreitol. In contrast to the enzymes that employ pyridoxal 5 -phosphate, detailed physical and mechanistic characterization of the iron-dependent deaminases is limited, primarily because of their extreme instability. We report here the characterization of L-serine deaminase from Escherichia coli, which is the product of the sdaA gene. When purified anaerobically, the isolated protein contains 1.86 ؎ 0.46 eq of iron and 0.670 ؎ 0.019 eq of sulfide per polypeptide and displays a UV-visible spectrum that is consistent with a [4Fe-4S] cluster. Reconstitution of the protein with iron and sulfide generates considerably more of the cluster, and treatment of the reconstituted protein with dithionite gives rise to an axial EPR spectrum, displaying gʈ ‫؍‬ 2.03 and g Ќ ‫؍‬ 1.93. Mö ssbauer spectra of the 57 Fe-reconstituted protein reveal that the majority of the iron is in the form of [4Fe-4S] 2؉ clusters, as evidenced by the typical Mö ssbauer parameters-isomer shift, ␦ ‫؍‬ 0.47 mm/s, quadrupole splitting of ⌬E Q ‫؍‬ 1.14 mm/s, and a diamagnetic (S ‫؍‬ 0) ground state. Treatment of the dithionitereduced protein with L-serine results in a slight broadening of the feature at g ‫؍‬ 2.03 in the EPR spectrum of the protein, and a dramatic loss in signal intensity, suggesting that the amino acid interacts directly with the cluster.

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Section: Discussionsupporting

confidence: 61%

Escherichia coli L-Serine Deaminase Requires a [4Fe-4S] Cluster in Catalysis

Cicchillo¹,

Baker²,

Schnitzer³

et al. 2004

Journal of Biological Chemistry

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“…We chose the EXAFS of k ranging from 3.12 to 12.00 Å Ϫ1 for analysis (Fig. 4B, inset) and applied the Fourier transform to yield the distances of the ligating atoms (iron and sulfur; (27,28). The EXAFS results were in good agreement with the EPR results, and a higher coordinated number of Fe-Fe bonds excluded the [2Fe-2S], supporting the temperature-dependent EPR results.…”

Section: Spectrophotometric Evidence Suggests a [2fe-2s] Or [4fe-4s] supporting

confidence: 60%

“…Following baseline correction (with Rbkg ϭ 1 using AUTOBK), the EXAFS data were analyzed and simulated using ARTEMIS to yield the fitting curves (24). Scattering paths of the [4Fe-4S] cluster were generated with the ATOMS (26) and FEFF (25) programs, in which the initial distance of Fe-Fe was set as 2.7 Å and that of Fe-S as 2.2 Å, based on measurements from the synthetic (27) and experimental Fe-S clusters (28). Two-shell models centered on iron atoms surrounded by (i) iron and sulfur atoms, such as [4Fe-4S]-(S-Cys) 4 and [3Fe-4S]-(S-Cys) 3 , or (ii) iron, sulfur, and oxygen, [4Fe-4S]-(O-Ser) 1 -(SCys) 3 , were used in data analysis, and only atoms at distances within 1.8 Յ R Յ 3.3 Å were considered.…”

Section: Chemicalsmentioning

confidence: 99%

FeoC from Klebsiella pneumoniae Contains a [4Fe-4S] Cluster

Hsueh

Chen

et al. 2013

J Bacteriol

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Iron is essential for pathogen survival, virulence, and colonization. Feo is suggested to function as the ferrous iron (Fe 2؉ ) transporter. The enterobacterial Feo system is composed of 3 proteins: FeoB is the indispensable component and is a large membrane protein likely to function as a permease; FeoA is a small Src homology 3 (SH3) domain protein that interacts with FeoB; FeoC is a winged-helix protein containing 4 conserved Cys residues in a sequence suitable for harboring a putative iron-sulfur (Fe-S) cluster. The presence of an iron-sulfur cluster on FeoC has never been shown experimentally. We report that under anaerobic conditions, the recombinant Klebsiella pneumoniae FeoC (KpFeoC) exhibited hyperfine-shifted nuclear magnetic resonance (NMR) and a UV-visible (UV-Vis) absorbance spectrum characteristic of a paramagnetic center. The electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) results were consistent only with the [4Fe-4S] clusters. Substituting the cysteinyl sulfur with oxygen resulted in significantly reduced cluster stability, establishing the roles of these cysteines as the ligands for the Fe-S cluster. When exposed to oxygen, the [4Fe-4S] cluster degraded to [3Fe-4S] and eventually disappeared. We propose that KpFeoC may regulate the function of the Feo transporter through the oxygen-or iron-sensitive coordination of the Fe-S cluster.

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“…The resulting short-lived ketyl radical would then represent a significantly stronger reductant, given the reduction potential of −1:5 V for a ketone vs. −0:9 V for the corresponding conjugate acid (24)(25)(26). Thus, the deprotonation of 8 to 9 could facilitate outer-sphere electron transfer from the radical intermediate to reduce the oxidized [4Fe-4S] 2+ cluster, which has a midpoint potential of around −1 V in aprotic solvents (27)(28)(29). Nevertheless, a much more positive reduction potential (−0:5 V) has been measured for the [4Fe-4S] 2+ cluster within the active site of the radical SAM enzyme lysine-2,3-aminomutase (30).…”

mentioning

confidence: 99%

EPR-kinetic isotope effect study of the mechanism of radical-mediated dehydrogenation of an alcohol by the radical SAM enzyme DesII

Ruszczycky

Choi

Liu

2013

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

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The radical S-adenosyl-L-methionine enzyme DesII from Streptomyces venezuelae is able to oxidize the C3 hydroxyl group of TDP-D-quinovose to the corresponding ketone via an α-hydroxyalkyl radical intermediate. It is unknown whether electron transfer from the radical intermediate precedes or follows its deprotonation, and answering this question would offer considerable insight into the mechanism by which the small but important class of radical-mediated alcohol dehydrogenases operate. This question can be addressed by measuring steady-state kinetic isotope effects (KIEs); however, their interpretation is obfuscated by the degree to which the steps of interest limit catalysis. To circumvent this problem, we measured the solvent deuterium KIE on the saturating steady-state concentration of the radical intermediate using electron paramagnetic resonance spectroscopy. The resulting value, 0:22 ± 0:03, when combined with the solvent deuterium KIE on the maximum rate of turnover (V) of 1:8 ± 0:2, yielded a KIE of 8 ± 2 on the net rate constant specifically associated with the α-hydroxyalkyl radical intermediate. This result implies that electron transfer from the radical intermediate does not precede deprotonation. Further analysis of these isotope effects, along with the pH dependence of the steady-state kinetic parameters, likewise suggests that DesII must be in the correct protonation state for initial generation of the α-hydroxyalkyl radical. In addition to providing unique mechanistic insights, this work introduces a unique approach to investigating enzymatic reactions using KIEs.T he enzyme-catalyzed dehydrogenation of an alcohol to a carbonyl is one of the most common and fundamental reactions of both primary metabolism and secondary metabolism. In the majority of cases studied, these reactions proceed via hydride transfer to an organic cofactor, such as the nicotinamide moiety of NAD(P) + or the isoalloxazine group of FAD among others (1, 2). In contrast, much less is understood regarding radical-mediated dehydrogenation of an alcohol, because only a handful of enzyme examples have been described. Of the radical alcohol dehydrogenases, galactose oxidase is the most extensively studied, and it is known to use a Cu II ion coordinated to a proteinderived 3′-(S-cysteinyl)tyrosyl radical to catalyze dehydrogenation of the C6 hydroxyl group of galactose (3, 4). In contrast, BtrN and the anaerobic sulfatase maturating enzymes (anSMEs) are radical S-adenosyl-L-methionine (SAM) enzymes (5-7) responsible for the dehydrogenation of the C3 hydroxyl group of 2-deoxy-scylloinosamine (8, 9) and the hydroxyl/sulfhydryl moieties of serine/ cysteine residues (10-12), respectively. In addition to the primary [4Fe-4S] 1+ cluster, which reduces SAM (1) to methionine (2) and the 5′-deoxyadenosyl radical (17; see Fig. 4) to initiate radical catalysis, BtrN (13,14) and anSMEs (15-18) possess one or more auxiliary [4Fe-4S] aux clusters, which have been proposed to coordinate the hydroxyl group to be oxidized in the substrate.The enzyme...

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Synthetic Analogues of the Active Sites of Iron−Sulfur Proteins

Cited by 520 publications

References 343 publications

Escherichia coli L-Serine Deaminase Requires a [4Fe-4S] Cluster in Catalysis

Escherichia coli L-Serine Deaminase Requires a [4Fe-4S] Cluster in Catalysis

FeoC from Klebsiella pneumoniae Contains a [4Fe-4S] Cluster

EPR-kinetic isotope effect study of the mechanism of radical-mediated dehydrogenation of an alcohol by the radical SAM enzyme DesII

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