Quatenary structure of methemoglobin II. Pulse radiolysis study of the binding of oxygen to the valence-hybrid. Progress report, December 1, 1978-November 30, 1979

Chevion, Mordechai; Ilan, Yigal; Samuni, Amram; Navok, T; Czapski, Gidon

doi:10.2172/6137131

Cited by 5 publications

(20 citation statements)

References 7 publications

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“…Similar results were observed previously for human methemoglobin [12] and fetal methemoglobin (unpublished results). In the absence of inositoLP6 at pH 7.8 and in the presence of inositol-P6 at pH 7.85, the analysis of the kinetic curves proved that the oxygenation is a single-phase reaction.…”

Section: Oxidation Of Myoglobin By Ferricyanidesupporting

confidence: 92%

“…For the stripped methemoglobin the values are pK 6.6 and h = 1.0, while in the presence of inositol-P6 pK = 6.9 and h = 1.2. Note that in a previous study with human methemoglobin [12] and in a study with fetal methemoglobin (unpublished), the absorption of the fastreacting species was significantly higher. Subsequently we repeated the calculations of the pK values and the Hill coefficient neglecting the assumption of the spectral identity of the fast and slow reacting species.…”

Section: Methodsmentioning

confidence: 59%

“…Under our experimental conditions less than 5 of the valence-intermediates are reduced in more than one site [12]. The reduction process is followed by spectral changes in the microsecond time range [13,15,16].…”

Section: Methodsmentioning

confidence: 96%

“…Below pH 7.8 the reaction was biphasic, and a computer 'best fit' to Eqn (3) was used to evaluate the parameters of the reaction : izing the fast and slow reacting species, respectively. The results, which are based on the analysis of biphasic kinetic curves described in detail previously [12], are summarized in Table 1. Above pH 7.8 only one phase is observed with a rate constant k = 7 .…”

Section: Methodsmentioning

confidence: 99%

“…3 and 2) shows that inositoLP6 increases the fraction of the T structure (i.e. stabilizes the hemoglobin in the low-affinity state [11,12]). …”

Section: Methodsmentioning

confidence: 99%

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Chain Inequivalence in Bovine Methemoglobin

Ilan

Chevion

Czapski

1980

European Journal of Biochemistry

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Using pulse radiolysis, a single heme in the tetramer of bovine methemoglobin was reduced within a few microseconds to the ferro state, producing a valence intermediate. The kinetics of oxygen binding to the valence intermediate as well as the re-oxidation of the ferro-heme to the ferric state were studied as a function of pH.The kinetics of the oxygenation revealed the existence of two species, characterized by high and low affinities for oxygen that are associated with two quaternary structures (R and T, respectively). A sigmoidal curve representing a transition between the two states as a function of pH was derived.Above pH 7.7 only the R state could be observed, while below pH 6.5 the T state was dominant.The reaction between the valence intermediate and ferricyanide at pH 7.75 (R state) consisted of two (about) equal contributions (kl = 23 x lo4 M-' s-'; k2 = 2.1 x lo4 M-' s-' ) attributed to the p and a subunits within the tetramer, respectively. At pH 6.3 (T state) a similar phenomenon was [4] also revealed that p chains are more reactive towards various ligands than a chains. In this investigation we have studied the oxidation of partly reduced bovine methemoglobin by ferricyanide and revealed chain inequivalence.Oxidation of human hemoglobin by ferricyanide has been studied by Antonini et al.[lo] using a stoppedflow apparatus. They found that below pH 8 the rate of oxidation is reduced as the reaction proceeds and they attributed this phenomenon to a possible intramolecular heterogeneity between a and p chains.The oxidation of tetrameric hemoglobin with ferricyanide is widely used for the preparation of methemoglobin. The reaction proceeds by a single electron transfer from the heme iron to ferricyanide producing oxidized ferric heme. Although the overall reaction has been studied in great detail [6] less information is available concerning the oxidation of individual chains within the tetramer. It is evident that non-equivalence of the subunits will be observed most clearly when the hemoglobin molecules are either fully in the high-affinity state or all in the lowaffinity state.The methemoglobin molecule is visualized as residing in either of two sets of quaternary structures, in each set the structures are closely related. In recent studies [ l l , 121 we have suggested that by using the pulse radiolysis technique the quaternary structure of methemoglobin can be characterized by following the kinetics of CO and O2 binding to its valence intermediate. The rate constants for these reactions were found to be dependent upon the pH and the presence of organic phosphates [ll, 121. The changes in these rate constants indicated a transition between affinity states representing a switch between quaternary structures.In this study the affinity state of bovine methemoglobin is monitored by studying the oxygenation of its valence intermediate. The changes of the oxygenation process enable us to characterize the conditions at which the methemoglobin molecule resides in either

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Section: Oxidation Of Myoglobin By Ferricyanidesupporting

confidence: 92%

Section: Methodsmentioning

confidence: 59%

Section: Methodsmentioning

confidence: 96%

Section: Methodsmentioning

confidence: 99%

“…3 and 2) shows that inositoLP6 increases the fraction of the T structure (i.e. stabilizes the hemoglobin in the low-affinity state [11,12]). …”

Section: Methodsmentioning

confidence: 99%

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Chain Inequivalence in Bovine Methemoglobin

Ilan

Chevion

Czapski

1980

European Journal of Biochemistry

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Conformational changes in hemoglobin triggered by changing the iron charge

et al. 2008

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In this work the hemoglobin conformational changes induced by changing the iron charge have been studied and compared with Myoglobin. Mössbauer spectroscopy was used to follow the change of the iron conformation. In order to compare the conformational relaxation of hemoglobin and myoglobin, and to study a possible influence of the quaternary structure, an intermediate metastable state of hemoglobin has been created by low temperature X-ray irradiation of methemoglobin. The irradiation reduces the Fe(III) of the heme groups to Fe(II) Low Spin, where the water is still bound on the sixth coordination. Heating cycles performed at temperatures from 140 K to 200 K allow the molecules to overcome an activation energy barrier and to relax into a stable conformation such as deoxyhemoglobin or carboxy-hemoglobin, if CO is present. Slightly different structures (conformational substates) reveal themselves as a distribution of energy barriers ( G # ). The distribution of the activation energy, for the decay of the Fe(II) Low Spin intermediate, has been fitted with a Gaussian. For comparison, published myoglobin data were re-analysed in the same way. The average energy value at characteristic temperature is very similar in case of myoglobin and hemoglobin. The larger Gaussian energy distribution for myoglobin with respect to hemoglobin shows that more conformational substates are available. This may be caused by a larger area exposed to water. In hemoglobin, part of the surface of the chains is not water accessible due to the quaternary structure.

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Reaction Intermediates of Nitric Oxide Synthase from Deinococcus radiodurans as Revealed by Pulse Radiolysis: Evidence for Intramolecular Electron Transfer from Biopterin to Fe^II–O₂ Complex

et al. 2018

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Nitric oxide synthase (NOS) is a cytochrome P450-type mono-oxygenase that catalyzes the oxidation of l-arginine (Arg) to nitric oxide (NO) through a reaction intermediate N-hydroxy-l-arginine (NHA). The mechanism underlying the reaction catalyzed by NOS from Deinococcus radiodurans was investigated using pulse radiolysis. Radiolytically generated hydrated electrons reduced the heme iron of NOS within 2 μs. Subsequently, ferrous heme reacted with O to form a ferrous-dioxygen intermediate with a second-order rate constant of 2.8 × 10 M s. In the tetrahydrofolate (HF)-bound enzyme, the ferrous-dioxygen intermediate was found to decay an another intermediate with a first-order rate constant of 2.2 × 10 s. The spectrum of the intermediate featured an absorption maximum at 440 nm and an absorption minimum at 390 nm. In the absence of HF, this step did not proceed, suggesting that HF was reduced with the ferrous-dioxygen intermediate to form a second intermediate. The intermediate further converted to the original ferric form with a first-order rate constant of 4 s. A similar intermediate could be detected after pulse radiolysis in the presence of NHA, although the intermediate decayed more slowly (0.5 s). These data suggested that a common catalytically active intermediate involved in the substrate oxidation of both Arg and NHA may be formed during catalysis. In addition, we investigated the solvent isotope effects on the kinetics of the intermediate after pulse radiolysis. Our experiments revealed dramatic kinetic solvent isotope effects on the conversion of the intermediate to the ferric form, of 10.5 and 2.5 for Arg and NHA, respectively, whereas the faster phases were not affected. These data suggest that the proton transfer in DrNOS is the rate-limiting reaction of the intermediate with the substrates.

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Quatenary structure of methemoglobin II. Pulse radiolysis study of the binding of oxygen to the valence-hybrid. Progress report, December 1, 1978-November 30, 1979

Cited by 5 publications

References 7 publications

Chain Inequivalence in Bovine Methemoglobin

Chain Inequivalence in Bovine Methemoglobin

Conformational changes in hemoglobin triggered by changing the iron charge

Reaction Intermediates of Nitric Oxide Synthase from Deinococcus radiodurans as Revealed by Pulse Radiolysis: Evidence for Intramolecular Electron Transfer from Biopterin to Fe^II–O₂ Complex

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Quatenary structure of methemoglobin II. Pulse radiolysis study of the binding of oxygen to the valence-hybrid. Progress report, December 1, 1978-November 30, 1979

Cited by 5 publications

References 7 publications

Chain Inequivalence in Bovine Methemoglobin

Chain Inequivalence in Bovine Methemoglobin

Conformational changes in hemoglobin triggered by changing the iron charge

Reaction Intermediates of Nitric Oxide Synthase from Deinococcus radiodurans as Revealed by Pulse Radiolysis: Evidence for Intramolecular Electron Transfer from Biopterin to FeII–O2 Complex

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Reaction Intermediates of Nitric Oxide Synthase from Deinococcus radiodurans as Revealed by Pulse Radiolysis: Evidence for Intramolecular Electron Transfer from Biopterin to Fe^II–O₂ Complex