The nature of the active site of hemerythrin

Klotz, Irving M.; Klotz, Themis A.; Fiess, Harold A.

doi:10.1016/0003-9861(57)90360-0

Cited by 112 publications

(55 citation statements)

References 14 publications

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“…to previous reports (4,22). Hemerythrin was isolated and purified from Phascolopsis gouldii as described previously (23,24). O 2 concentrations were determined by using a YSI 5300 biological oxygen monitor (Yellow Springs Instruments, Yellow Springs OH).…”

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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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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Section: Discussionmentioning

confidence: 99%

“…Hemerythrin is known to contain a substantial amount of iron (Klotz et al 1957), so that an iron-sulfide interaction seems likely. In contrast to hemoglobin, hemerythrin binds oxygen by changing its valence from Fe2+ to ~e~+ (Klotz et al 1957, Klippenstein 1980. Our preliminary studies on the coelomic fluid show that the black component is associated in the erythrocytes and has a molecular weight > 10 kDa, when we used a centrifugation procedure that excludes molecules of this size.…”

Section: Discussionmentioning

confidence: 99%

Sulfide detoxification and tolerance in Halicryptus spinulosus (Priapulida): a multiple strategy

Oeschger¹,

Vetter²

1992

Mar. Ecol. Prog. Ser.

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“…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).…”

Section: Methodsmentioning

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 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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The nature of the active site of hemerythrin

Cited by 112 publications

References 14 publications

Oxygen Kinetic Isotope Effects in Soluble Methane Monooxygenase

Oxygen Kinetic Isotope Effects in Soluble Methane Monooxygenase

Sulfide detoxification and tolerance in Halicryptus spinulosus (Priapulida): a multiple strategy

Active site structures of deoxyhemerythrin and oxyhemerythrin.

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