Spectroscopy and structure of hemerythrin

Garbett, K.; Darnall, Dennis W.; Klotz, Irving M.; Williams, R. J. P.

doi:10.1016/0003-9861(69)90559-1

Cited by 128 publications

(124 citation statements)

References 50 publications

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“…The exogenous ligand binding site is vacant in methemerythrin, with a compensating shift towards trigonal bipyramidal coordination for the five ligands of Fe2. The observation of an oxo-bridged binuclear iron structure in both forms of the protein is consistent with the magnetic and spectroscopic evidence for two strongly coupled high-spin Fe(III) atoms in the metal complex in methemerythrins (5,6).…”

supporting

confidence: 85%

“…Even at this resolution, however, the crystallographic results are valuable because of the limited structural information for deoxyhemerythrin currently available from other techniques. Mossbauer spectroscopy indicates that the complex comprises two high-spin Fe(II) atoms (5,7), and magnetic susceptibility measurements show little antiferromagnetic coupling between the Fe atoms (5,8). The absence of a distinct visible absorption spectrum for deoxyhemerythrin has hampered spectroscopic investigations of the Fe complex in this form of the protein.…”

mentioning

confidence: 99%

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Active site structures of deoxyhemerythrin and oxyhemerythrin.

Stenkamp¹,

Sieker²,

Jensen³

et al. 1985

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

136

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The physiologically active forms of the nonheme-iron, oxygen-transport protein hemerythrin have been studied by x-ray crystallographic techniques. At 3.9-A resolution, a difference electron-density map between the deoxy form and met form (methemerythrin) of the protein suggests only small differences in the binuclear iron complexes. The coordination of the iron atoms appears to be the same in both the deoxy and met forms, one iron of the complexes being pentacoordinate, the other iron being hexacoordinate. The iron atoms appear to be somewhat farther apart in the deoxy form. A 2.2-A resolution study of oxyhemerythrin shows that dioxygen binds to one iron atom-the pentacoordinate one in the met form of the protein, the same binding site found for azide in azidomethemerythrin.The initial objective of the crystallographic structure determinations of hemerythrin was to structurally characterize this nonheme-iron, oxygen-transport protein to provide a basis for understanding the chemical differences between its derivatives. The early studies focused on the structure of the oxidized form, or methemerythrins (1, 2), primarily because they are more stable than are the physiologically more interesting deoxy and oxy forms. The active center is a binuclear iron complex bound to the protein by amino acid side chains. In azidomethemerythrin, the iron atoms are both hexacoordinate ( Fig. 1 Left). In the met form [formerly identified as aquomet (3) or hydroxomet (4)], one of the iron atoms, designated Fe2, is pentacoordinate ( Fig. 1 (11), and x-ray absorption spectroscopy (12) indicate that the metal complex in oxyhemerythrin is similar to that in azidomethemerythrin, where the two Fe(III) atoms are bridged by a A-oxygen atom, and the exogenous ligand binds only to one Fe atom. The x-ray crystallographic results reported here support this description for oxyhemerythrin and, in addition, suggest some rearrangement in the binuclear iron complex upon conversion from deoxy to oxy. The 2.2-A resolution crystallographic investigations provide important structural information bearing on the functional properties of the protein and limiting the mechanisms to be considered in explaining the biological action of the molecule. EXPERIMENTALThe sipunculid T. dyscrita was obtained from the Oregon Institute of Marine Biology (Charleston, OR). Hemerythrin was extracted and crystallized from the coelomic fluid as described (13, 14). The crystals were dissolved in 0.2 M Tris sulfate (pH 8.0) and concentrated to -30 mg of protein per ml by ultrafiltration (XM-50 filter; Amicon). The hemerythrin solution was depleted of oxygen by anaerobic dialysis against nitrogen-purged buffer. A 4-fold molar excess (per hemerythrin monomer) of solid sodium dithionite was added to the dialysis buffer. After 25 hr the protein solution displayed the pale yellow color characteristic of reduced hemerythrin. Crystals of deoxyhemerythrin were obtained by slow dialysis of the concentrated solution against 0.02 M Tris sulfate (pH 7.2) under anaerobic condition...

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supporting

confidence: 85%

mentioning

confidence: 99%

Active site structures of deoxyhemerythrin and oxyhemerythrin.

Stenkamp¹,

Sieker²,

Jensen³

et al. 1985

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

136

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“…The yield, based on ligand, was only 27%. Its visible light spectrum consisted of a similar MLCT band at 369 nm (E 1469 M --' cm-') and a much broader d-d transition centred near 631 nm (E 127 M-' cm-'), similar to that shown by met-chloro hemerythrin (656 nm, E 180 M -I cm-I) (25). Though this was not the exclusive product (there was an insoluble brown material produced here as well), the tendency for the metal to bind two ligands was a problem here again.…”

Section: : X -C Hmentioning

confidence: 74%

Short syntheses of rigid acyclic or macrocyclic poly-imine ligands and their use in Ru(II), Fe(III), and alkali metal complexation

Dash¹,

Potvin²

1992

Can. J. Chem.

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ROBERT DASH and PIERRE G. POTVIN. Can. J. Chem. 70, 2249 (1992. Lipophilic derivatives of 2,6-bis(3-pyrazoly1)pyridine and 1,3-bis (3-pyrazoly1)benzene were obtained in acceptable yields by Stork reactions and transformed by N-alkylations into three pentadentates, three bis(bidentates), and one macrocyclic octadentate ligand. The parent bis(pyrazoly1) pyridine constituted a terpyridine analogue and readily formed a Ru(I1) complex. By comparison of 'H nuclear magnetic resonance patterns, it was found that the alkylations in the bis(pyrazoly1)pyridine series conferred an anti,anti conformation of the imino nitrogens. The pentadentates were then able to extract alkali metal ions into CHCI, with only modest strength and selectivity, but the preferred extraction of Li' by one of these was notable. By variation of the reaction conditions, a tri-iron(II1) and a di-iron(II1) complex were obtained from a bis(bidentate).ROBERT DASH et PIERRE G. POTVIN. Can. J. Chem. 70, 2249Chem. 70, (1992. Des derives lipophiles de la 2,6-bis(3-pyrazo1yle)pyridine et du 1,3-bis(3-pyrazoly1e)benzkne furent prepares en rendements acceptables par des reactions de Stork, puis N-alkylks pour fournir trois pentadentates, trois bis(bidentate) et un octadentate macrocyclique. En tant qu'analogue de la terpyridine, la bis(pyrazolyle)pyridine forma aisement un complexe au Ru(I1). D'aprks la resonance magnetique nuclkaire, les alkylations de la bis(pyrazoly1e)pyridine ont force l'adoption de conformations anti,anti et les pentadentates etaient donc de modestes recepteurs pour les cations alkalins et ont demontrk une faible sClectivit6 en extraction dans le chloroforme. Neanmoins, on souligne l'extraction preferentielle du Li' par l'un d'eux. Un bis(bidentate1 forma, selon les conditions employees, des complexes bi-et tri-nuclkaires avec le Fe(II1).

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“…Deoxyhemerythrin was prepared by reducing hemerythrin with excess sodium dithionite, followed by two dialysis steps against anaerobic 30 mM Tris-SO 4 , pH 8.2. Upon aeration, this preparation showed the optical spectrum typical of oxyhemerythrin (A 330 nm /A 500 nm ϭ 3.2), and no significant amount of methemerythrin (25). In the binding studies, a 150-l aliquot of anaerobic deoxyhemerythrin (283 M) in 30 mM Tris-SO 4 , pH 8.2 was added to 1.5 ml of air-saturated 30 mM Tris-SO 4 , pH 8.…”

Section: Methodsmentioning

confidence: 99%

Oxygen Kinetic Isotope Effects in Soluble Methane Monooxygenase

Stahl

Francisco

Merkx

et al. 2001

Journal of Biological Chemistry

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Soluble methane monooxygenase (sMMO) contains a nonheme, carboxylate-bridged diiron site that activates dioxygen in the catalytic oxidation of hydrocarbon substrates. Oxygen kinetic isotope effects (KIEs) have been determined under steady-state conditions for the sMMO-catalyzed oxidation of CH 3 CN, a liquid substrate analog. Kinetic studies of the steady-state sMMO reaction revealed a competition between fully coupled oxygenase activity, which produced glycolonitrile (HOCH 2 CN) and uncoupled oxidase activity that led to water formation. The oxygen KIE was measured independently for both the oxygenase and oxidase reactions, and values of 1.0152 ؎ 0.0007 and 1.0167 ؎ 0.0010 were obtained, respectively. The isotope effects and separate dioxygen binding studies do not support irreversible formation of an enzyme-dioxygen Michaelis complex. Additional mechanistic implications are discussed in the context of previous data obtained from single turnover and steady-state kinetic studies.The soluble methane monooxygenase system (sMMO) 1 found in certain methanotrophic bacteria catalyzes the oxidation of methane to methanol according to Reaction 1 (1, 2).This reaction is the first essential step of a metabolic pathway that allows these bacteria to use methane as their sole source of energy and carbon. The soluble MMO system comprises three protein components: a 251 kDa hydroxylase (MMOH), a reductase (MMOR) that transfers electrons from NADH to MMOH, and a cofactorless regulatory protein (MMOB). Reductive activation of dioxygen and subsequent methane oxidation take place at a carboxylate-bridged diiron cluster in the active site of MMOH. (3,4,6,8). This observation suggests that Q, or a subsequent unobserved intermediate, is responsible for methane oxidation.Recent kinetic studies and theoretical calculations have suggested the presence of additional intermediates (6, 9, 10). For example, proton transfer steps have been observed in the formation and decay of H peroxo , a result consistent with a protonated peroxo intermediate in the mechanism (9). Other kinetic studies provide indirect evidence for one or more intermediates preceding the formation of H peroxo . These may include an enzyme-dioxygen Michaelis complex or a superoxo intermediate (6, 9 -11).Kinetic and equilibrium oxygen isotope effects have been determined recently for several proteins and enzymes that activate dioxygen (12)(13)(14)(15)(16)(17)(18). Such experiments directly probe reaction steps involved in the activation of dioxygen and can provide unique mechanistic information that may not be available by other means. In this study, oxygen-18 kinetic isotope effects determined for sMMO from M. capsulatus (Bath) together with steady-state kinetics and dioxygen binding studies yield further insights into dioxygen activation by this enzyme system. EXPERIMENTAL PROCEDURESGeneral Considerations-Growth of M. capsulatus (Bath) cells and subsequent purification of MMOH were carried out as described previously (19,20). Both MMOB and MMOR were obtained from recombi...

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Spectroscopy and structure of hemerythrin

Cited by 128 publications

References 50 publications

Active site structures of deoxyhemerythrin and oxyhemerythrin.

Active site structures of deoxyhemerythrin and oxyhemerythrin.

Short syntheses of rigid acyclic or macrocyclic poly-imine ligands and their use in Ru(II), Fe(III), and alkali metal complexation

Oxygen Kinetic Isotope Effects in Soluble Methane Monooxygenase

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