Electronic Absorption, EPR, and Resonance Raman Spectroscopy of CooA, a CO-Sensing Transcription Activator from <i>R. rubrum</i>, Reveals a Five-Coordinate NO-Heme

Reynolds, Mark F.; Parks, Ryan B.; Burstyn, Judith N.; Shelver, Daniel; Thorsteinsson, Marc V.; Kerby, Robert L.; Roberts, Gary P.; Vogel, Kathleen M.; Spiro, Thomas G.

doi:10.1021/bi991378g

Cited by 93 publications

(124 citation statements)

References 65 publications

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“…8) [66]. These simulated values are in good agreement with those of other 5c {FeNO} hemoproteins [63,64,[67][68][69][70]. No spectral changes were observed with changes in pH (data not shown).…”

Section: Fe(ii) Rhed Binds No(g)supporting

confidence: 82%

CO and NO bind to Fe(II) DiGeorge critical region 8 heme but do not restore primary microRNA processing activity

et al. 2016

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Section: Fe(ii) Rhed Binds No(g)supporting

confidence: 82%

CO and NO bind to Fe(II) DiGeorge critical region 8 heme but do not restore primary microRNA processing activity

et al. 2016

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“…Six-coordinate S ϭ 1͞2 {FeNO} 7 species in heme proteins have much stronger Fe-NO bonds, as evidenced by (Fe-NO) frequencies in the range 536-558 cm Ϫ1 (20,21,41), and much weaker N-O bonds, as evidenced by (N-O) frequencies in the range 1,555-1,624 cm Ϫ1 (20,21). Although no clear consensus has emerged concerning the most appropriate description of the electronic structure and bonding in S ϭ 1͞2 {FeNO} 7 species (18,32,42), the increase in Fe-NO bond strength and concomitant decrease in the N-O bond strength, compared with S ϭ 3͞2 {FeNO} 7 species, appear to be a direct consequence of increased -donation into the empty d z2 orbital that results from lowering the Fe spin state from high to low or intermediate spin.…”

Section: Discussionmentioning

confidence: 99%

Nitric oxide binding at the mononuclear active site of reduced Pyrococcus furiosus superoxide reductase

Clay

Cosper

Jenney

et al. 2003

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

133

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O ver the past 4 yr, evidence has accumulated for a novel pathway for detoxification of reactive oxygen species that is specific for anaerobic and microaerophilic microorganisms (1-3). The key enzyme in this pathway is superoxide reductase (SOR), which catalyzes the reduction of superoxide to hydrogen peroxide (4), with rubredoxin as the probable physiological electron donor (2, 5). Although all SORs have a ␤-barrel domain containing a unique type of mononuclear Fe center that serves as the site for superoxide reduction (6, 7), some have an additional N-terminal domain that contains an intrinsic rubredoxin-like mononuclear Fe site, which is ligated by four cysteines in a distorted tetrahedral arrangement analogous to that found in desulforedoxin (6). These two classes are conveniently referred to as 1Fe-and 2Fe-SORs, but they are also known by their trivial names, neelaredoxins and desulfoferrodoxins, respectively.Spectroscopic and crystallographic studies of the 1Fe-SOR from Pyrococcus furiosus (7-9) and the 2Fe-SOR from Desulfovibrio desulfuricans (6, 10, 11) have shown that the mononuclear iron active site is ligated by four equatorial histidines (3 N and 1␦N) and one axial cysteinate in a square-pyramidal arrangement. The sixth coordination site appears to be occupied by a monodentate glutamate in the oxidized state but is vacant or occupied by a weakly coordinated water molecule in the reduced state, thereby providing a site for superoxide binding and reduction. These structural studies, combined with recent mutagenesis and pulse radiolysis kinetic results (12-16), have led to the proposal for the catalytic mechanism shown in Fig. 1 Characterization of enzymatic intermediates is required for both detailed understanding of the mechanism of SOR and addressing the key questions of how and why the SOR active site preferentially catalyzes reduction rather than dismutation of superoxide. However, these intermediates are short lived and difficult to study experimentally. Nitric oxide (NO) has been extensively used as a substrate analog of molecular oxygen to form stable nitrosyl derivatives that provide insight into oxygen transport and activation intermediates in many heme (18-21) and nonheme (22-29) iron enzymes. Hence, we report here the formation and spectroscopic characterization of a stable NO-bound derivative of the reduced 1Fe-SOR from P. furiosus using the combination of EPR, UV-visible absorption, and variable-temperature, variable-field magnetic CD (VTVH MCD), resonance Raman, and Fourier transform IR (FTIR) spectroscopies. The structural and electronic characterization of the NO adduct of SOR facilitates understanding of how the active site is tuned for oxidative addition of superoxide and release rather than intraligand cleavage of the peroxide product. Materials and MethodsBiochemical Techniques and Sample Preparation. Recombinant natural abundance and 34 S globally enriched P. furiosus SOR was This paper was submitted directly (Track II) to the PNAS office.Abbreviations: SOR, superoxide reductase; V...

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“…The UV-visible spectra of CO-, imidazole-, and CN Ϫ -bound ⌬P3R4 CooA were obtained with high pH anisotropy buffer for proper comparison with DNA binding activities of these forms. has been demonstrated that other potential exogenous ligands cannot create the 6-coordinate CooA adduct; NO is the only small molecule, other than CO, that can form a stable adduct to the heme of CooA (16), but NO binding results in a 5-coordinate form that appears to be inactive (16). This has led to the hypothesis that CO specificity results from its unique ability to create a 6-coordinate heme in CooA.…”

Section: Methodsmentioning

confidence: 99%

“…Small molecules that are weaker ligands than CO fail to displace the Pro 2 ligand, whereas NO displaces both protein ligands and leads to an inactive protein (16), and O 2 binding oxidizes CooA. It was therefore a reasonable hypothesis that the specificity for CO simply reflected its liganding strength.…”

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confidence: 99%

The Role of the Hydrophobic Distal Heme Pocket of CooA in Ligand Sensing and Response

Youn

Kerby

Roberts

2003

Journal of Biological Chemistry

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CooA from Rhodospirillum rubrum is a heme-containing transcriptional activator that becomes activated only upon binding CO. The basis for this specificity has been probed in a CooA variant, termed ⌬P3R4 CooA, lacking two residues adjacent to the Pro 2 heme ligand, which weakens that ligand. ⌬P3R4 CooA can bind imidazole and CN ؊ , as well as CO, and form a 6-coordinate low spin adduct with each. However, in contrast to the case with CO, imidazole and CN ؊ do not stimulate the DNA binding activity of ⌬P3R4 CooA. This result indicates that the CO-specific activation of CooA is not merely the result of creation of a 6-coordinate CooA adduct but that there must be another element to this response. One feature of CooA activation is modest repositioning of the C-helices upon CO binding, so we altered a portion of the C-helix (residues Ile 113 and Leu 116 ) located near the heme-bound CO in wild type CooA, and we investigated the effect on CO-specific activation. Surprisingly, the sizes of Ile 113 and/or Leu 116 positions are not critical for CooA activation by CO, disproving a precise interaction between these residues and the CO-bound heme as a basis for the CO activation mechanism and CO ligand specificity. In contrast, hydrophobic residues at these positions contribute to the activation. Some CooA variants altered at these positions in the background of ⌬P3R4 were also found to show low but reproducible activation in response to imidazole binding to the heme. A model for the role of hydrophobicity in CooA activation and specificity is suggested.

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Electronic Absorption, EPR, and Resonance Raman Spectroscopy of CooA, a CO-Sensing Transcription Activator from R. rubrum, Reveals a Five-Coordinate NO-Heme

Cited by 93 publications

References 65 publications

CO and NO bind to Fe(II) DiGeorge critical region 8 heme but do not restore primary microRNA processing activity

CO and NO bind to Fe(II) DiGeorge critical region 8 heme but do not restore primary microRNA processing activity

Nitric oxide binding at the mononuclear active site of reduced Pyrococcus furiosus superoxide reductase

The Role of the Hydrophobic Distal Heme Pocket of CooA in Ligand Sensing and Response

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