Chemical and spectroscopic studies of the mixed-valent derivatives of the non-heme iron protein hemerythrin

McCormick, James M.; Reem, Richard C.; Solomon, Edward I.

doi:10.1021/ja00024a007

Cited by 49 publications

(56 citation statements)

References 2 publications

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“…However, these signals are characteristic of the partially reduced semi-met Hr from one-electron reduction of the met-hemerythrin. The sample of metHr as isolated from M. capsulatus (Bath) yielded an EPR spectrum with g at 1.90, 1.84 and 1.50, which was similar (the g-values are somewhat more isotropic here) to the EPR observed for the semi-met Hr in the case of the marine invertebrate hemerythrins [21][22][23]. EPR spin-counting indicated that the semi-met form accounted for no more than 22% of the as-isolated protein sample.…”

Section: The Diiron Coresupporting

confidence: 61%

Isolation, purification and characterization of hemerythrin from Methylococcus capsulatus (Bath)

Kao

Wang

Huang

et al. 2008

Journal of Inorganic Biochemistry

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Section: The Diiron Coresupporting

confidence: 61%

Isolation, purification and characterization of hemerythrin from Methylococcus capsulatus (Bath)

Kao

Wang

Huang

et al. 2008

Journal of Inorganic Biochemistry

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“…The g-tensors of organic radicals and high-spin Fe III ions are generally less anisotropic than is observed for the ground state of G. However, quantum mechanical mixing of excited spin states with the S ϭ 1͞2 ground state increases g-anisotropy, as has been described for valence-localized high-spin diiron(II͞III) complexes (26)(27)(28). For the hypothetical (superoxo)diiron(III͞III) complex, the extent of mixing would depend on the zero-field splitting parameters of the Fe III ions [D Fe(III) , (E͞D) Fe(III) ] and the energy gaps between the spin ground and excited states, which would be determined by the three J values.…”

Section: Discussionmentioning

confidence: 90%

Evidence for C–H cleavage by an iron–superoxide complex in the glycol cleavage reaction catalyzed by myo -inositol oxygenase

Xing¹,

Diao²,

Hoffart³

et al. 2006

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

110

167

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. Recent studies have suggested that the enzyme, which was shown nearly 50 years ago to require iron (1, 2), contains a coupled dinuclear nonheme iron cluster (5), making MIOX the most recent addition to the nonheme diiron oxygenase͞oxidase family that also includes bacterial hydrocarbon hydroxylases (e.g., soluble methane monooxygenase), plant fatty acyl desaturases (e.g., stearoyl acyl carrier protein ⌬ 9 desaturase), and protein R2 of class I ribonucleotide reductase (R2) (6-10). Mössbauer and EPR spectra showed that treatment of recombinant Mus musculus MIOX isolated in its iron-free form from Escherichia coli with Fe(II) and O 2 leads to formation of an antiferromagnetically coupled diiron cluster in either the II͞III or III͞III oxidation state, depending on the O 2 ͞MIOX ratio and the presence or absence of a reductant (ascorbate or cysteine). Binding of MI was shown to perturb the spectra of both oxidation states in a manner consistent with direct coordination of the substrate to the cluster (5).All nonheme diiron oxygenases and oxidases characterized before MIOX activate O 2 with the II͞II oxidation state of the cofactor (11,12). For several of the reactions, (peroxo)diiron(III͞III) intermediates have been demonstrated. These complexes are generally proposed to undergo O-O-bond cleavage to generate high-valent iron complexes that cleave strong C-H or O-H bonds of their oxidation targets (8,(11)(12)(13)(14). Indeed, the diiron(III͞IV) cluster, X (15, 16), and the diiron(IV͞IV) cluster, Q (8, 13, 17), have been directly characterized in the R2 and soluble methane monooxygenase reactions, respectively. In each of the previously characterized diiron-oxygenase͞oxidase reactions, a diiron(III͞III) ''product'' state of the cluster is generated at the end of the oxidation sequence. Subsequent events require reduction of the cluster back to the diiron(II͞II) state by additional proteins, with electrons provided ultimately by NAD(P)H. This redox cycling of the cofactor and provision of two electrons by the nicotinamide ''cosubstrate'' ensure that at most two electrons can be extracted from the substrate. The MIOX reaction, a four-electron oxidation, would seem to require a different mechanism.Indeed, a recent study concluded that the mixed-valent, II͞III state of the cofactor, rather than the conventional II͞II state, activates O 2 for DG production in the MIOX reaction (4). Single-turnover experiments showed that the fully reduced enzyme (MIOX II/II ) reacts with limiting O 2 in the presence of saturating MI to generate the mixed-valent enzyme as a stable product with unit stoichiometry and with only a low yield of DG. By contrast, the

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“…The EPR spectrum of the mixed-valence form of NorA with g value components Ͻ2.0 shows that NorA contains a di-iron center (47)(48)(49). Regarding the low amount of the mixed-valence signal, being highest in aerobically purified NorA, we consider the mixed-valence state as a non-physiological intermediate.…”

Section: Discussionmentioning

confidence: 99%

Formation of a Dinitrosyl Iron Complex by NorA, a Nitric Oxide-binding Di-iron Protein from Ralstonia eutropha H16

Strube

Vries

Cramm

2007

Journal of Biological Chemistry

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In Ralstonia eutropha H16, two genes, norA and norB, form a dicistronic operon that is controlled by the NO-responsive transcriptional regulator NorR. NorB has been identified as a membrane-bound NO reductase, but the physiological function of NorA is unknown. We found that, in a NorA deletion mutant, the promoter activity of the norAB operon was increased 3-fold, indicating that NorA attenuates activation of NorR. NorA shows limited sequence similarity to the oxygen carrier hemerythrin, which contains a di-iron center. Indeed, optical and EPR spectroscopy of purified NorA revealed the presence of a di-iron center, which binds oxygen in a similar way as hemerythrin. Nitric oxide (NO)2 is an obligate intermediate of denitrification (1, 2), formed by the reduction of nitrite by nitrite reductases and subsequently converted to nitrous oxide by NO reductases. In the denitrifier Ralstonia eutropha H16, formation of the NO reductase NorB is controlled by NorR, an NObinding transcriptional activator (3, 4). The norB gene is cotranscribed with norA that encodes a protein of unknown function. Orthologs of NorA are present in many genomes of proteobacteria and firmicutes (5) and have been annotated as DnrN (Pseudomonas stutzeri), ScdA (Staphylococcus aureus), or YtfE (Escherichia coli and Salmonella enterica). The expression of DnrN, ScdA, and YtfE has been shown to be up-regulated by NO or nitrosating agents (6 -9). The non-denitrifiers S. aureus, E. coli, and S. enterica, however, do not possess the norB gene, which demonstrates that norA-like genes do not necessarily co-occur with norB. The NO-dependent expression of norA and its coexpression with norB in R. eutropha suggest a function for NorA in NO metabolism. However, it was shown previously that a nonpolar in-frame deletion of the norA gene did not affect denitrification of R. eutropha cells in terms of growth, accumulation of nitrite or nitrous oxide, or formation of dinitrogen (4). In this study we report the purification and characterization of NorA from R. eutropha. We show that NorA is an NO-binding di-iron protein that modulates the NOresponsive expression of the norAB operon in denitrifying cells of R. eutropha. EXPERIMENTAL PROCEDURESStrains and Plasmids-Strains and plasmids used in this study are listed in Table 1. E. coli JM109 was used for propagation of plasmids. E. coli S17-1 served as the donor in conjugative transfers. E. coli BL21(DE3) was used for overproduction of NorAhexahistidine fusion protein. Mutant strains of R. eutropha were constructed by a gene replacement strategy (15) using the suicide vector pLO1, and were verified by Southern blot analysis. The NorR, NorA, and NorB proteins referred to in this study are encoded by the norR1, norA1, and norB1 genes located on megaplasmid pHG1 of R. eutropha. A chromosomally encoded paralog nor gene cluster termed norR2A2B2 has not been extensively studied, but mutational analyses indicate that either of the two nor clusters is sufficient for denitrification (4).All R. eutropha strains used in this stu...

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Chemical and spectroscopic studies of the mixed-valent derivatives of the non-heme iron protein hemerythrin

Cited by 49 publications

References 2 publications

Isolation, purification and characterization of hemerythrin from Methylococcus capsulatus (Bath)

Isolation, purification and characterization of hemerythrin from Methylococcus capsulatus (Bath)

Evidence for C–H cleavage by an iron–superoxide complex in the glycol cleavage reaction catalyzed by myo -inositol oxygenase

Formation of a Dinitrosyl Iron Complex by NorA, a Nitric Oxide-binding Di-iron Protein from Ralstonia eutropha H16

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