Abel Schejter

Binding of hydrogen donors to horseradish peroxidase: a spectroscopic study.

Absorption spectroscopy measurements of the binding of aromatic donors and competitive inhibitors to horseradish peroxidase indicate that they are bound to the enzyme through hydrophobic forces and hydrogen bonding. Nuclear magnetic resonance experiments show that the minimal distances between the enzyme iron and the protons of a typical donor, p-cresol, are 7.0 ± 0.5, 7.7 ± 0.5 and 8.5 ± 0.5 A, for the ortho-, meta- and methyl-protons, respectively. A model for the binding of aromatic donors to horseradish peroxidase based on this result is presented. It is proposed that the aromatic ring is attached to a hydrophobic region in the protein interior and the phenol oxygen is hydrogen-bonded to the pyrrolic nitrogen of the iron-coordinated histidine. This structure is compatible with the proton-iron distances measured and offers an intramolecular path for electron conduction from donor to heme analogous to that proposed by Winfield for the peroxidases.

Nuclear magnetic resonance evidence for the absence of iron coordinated water in horseradish peroxidase

Summary The pH dependence of longitudinal relaxation rates of water protons in horseradish peroxidase solutions indicates that water is not coordinated to the ferric iron of the enzyme.

Preparation and properties of crosslinked water-insoluble catalase

Abstract Catalase was crosslinked to a water-insoluble network by treatment with a bifunctional reagent, glutardialdehyde. The catalatic activity of water-insoluble catalase was 10 times less than for the soluble native enzyme. The peroxidatic activity with methanol as hydrogen donor decreased 11-fold, while the decrease was 80-fold when the donor tested was pyrogallol. The insoluble enzyme was stable for several months at 4 °. Catalase inhibitors, such as cyanide, hydroxylamine, and aminotriazole, affect the insoluble and the soluble enzyme similarly. Cyanide also causes the same spectral changes in the insoluble catalase as those found in the soluble enzyme.

Structural studies of modified cytochromes c by nuclear magnetic resonance spectroscopy

Abstract The binding of methionine to one of the axial positions of the heme iron in various modified mammalian-type cytochromes c has been studied by nuclear magnetic resonance spectroscopy. The modifications of the protein included formylation of tryptophan, alkylation of methionine, and polymerization by treatment with ethanol. Comparison of the data on methionine coordination with data on the ability of the modified cytochromes c to restore the respiration of cytochrome c -depleted rat liver mitochondria, indicates that the redox carrier properties of cytochrome c are maintained only if methionine is bound to the heme iron in the oxidized and the reduced form.

Thermodynamics of the redox reaction of cytochromes c of five different species

The redox potential of cytochrome c has interested many investigators, because of its possible significance in understanding both the biological role played by this molecule, and also the relationship between its structure and physicochemical properties [l] . A better insight into this relationship can be gained through knowledge of enthalpy and entropy changes of the redox reaction. Such data were not available until recent calorimetric determinations by George et al. [2] The evaluation of the heat change for several cytochromes c, made by applying the Van? Hoff isochore to equilibrium measurements at various temperatures, is reported in this paper.

Single step chromatographic purification and characterization of the endopolygalacturonases and pectinesterases of the fungus, Botrytis cinerea Pers

Abstract This communication describes the purification, in a single chromatographic step on cross-linked polypectate, of two pectinesterases and two endopolygalacturonases found inthe extracellular fluid of cultures of the fungus, Botrytis cinerea . The endopolygalacturonases, PG-I and PG-II, have molecular weights of 34 000 and 56 000, respectively. They differ in their chromatographic behaviour, PG-II being more strongly retained by the cross-linked polypectate column. The pectinesterases, PE-I and PE-II, have nearly identical molecular weights of about 28 000 and very similar amino acid compositions, suggesting a common genetic origin. It is shown that Michaelis-Menten behaviour is obeyed by all these enzymes. The enzyme kinetic constants of PG-I and PG-II are strongly dependent on the salt concentration of the medium; both polyglutamate and pectin act as competitive inhibitors. The properties of PE-I and PE-II of B. cinerea are found to be strikingly similar to those of the two pectin-esterases purified from oranges.

The role of the lysines in the alkaline heme-linked ionization of ferric cytochrome c.

Abstract Fully amidinated cytochrome c has a 695 nm band which is lost with a pK of 9.2 in a transition to a low-spin state. Fully maleylated cytochrome c can also exist in a low-spin state without the 695 nm band although the pH of the transition is very dependent on the salt concentration. Neither of these derivatives have free lysine groups. It is concluded that the hemelinked alkaline ionization of native cytochrome c is not triggered by deprotonation of a lysine residue, and that lysine does not necessarily coordinate the iron in the low-spin alkaline species.

On the nature of the alkaline ionization of horseradish peroxidase.

It has been customary in hemoprotein research to attribute the alkaline ionization of high spin ferric hemoproteins to the proton dissociation of a11 iron bound water molecule [ 11 For methemoglobin and metmyoglobin, kinetic studies of their ionization with pK 8.8 have shown that, although the overall reaction is rather complicated. it includes a step that can indeed be interpreted as the ionization ofwater [L4]. Horseradish peroxidase in the ferric state undergoes an ionization with pK 10.8 that affects the redox potential of the enzyme [S] and results in a spectral change from a pure high-spin type to a type having mostly low-spin characteristics [6] By analogy, this ionization could also be considered to retlect the unprotonation of an iron-bound water molecule. However, the kinetics of this change, described in this paper, indicate that a conformation change of the molecule appears to be involved and suggest a different mechanism as responsible for the observed ionization.

Isolation and properties of the hemoglobin of the clam shrimp Cyzicus cf. hierosolymitanus (S. Fischer)

Abstract 1. 1. The blood hemoglobin of the conchostrac Cyzicus cf. hierosolymitanus has been isolated and purified. 2. 2. This hemoglobin has an apparent molecular weight of 2·2×10 5 12–13 subunits. 3. 3. The spectroscopic properties and amino acid composition are given. The oxygen equilibrium curve shows high oxygen affinity ( P 50 = 3·5 × 10 −2 mm Hg O 2 ) and indicates the presence of positive heme-heme interactions ( n = 2·3).

1H-NMR studies of the coordination geometry at the heme iron and the electronic structure of the heme group in cytochrome c-552 from Euglena gracilis

The 1H-NMR lines of heme c in reduced and oxidized cytochrome c-552 from Euglena gracilis were individually assigned and the coordination geometry of the axial ligands was investigated. The electronic structure of the heme and the chirality of the axially bound methionine were found to be of the same type as in mammalian cytochrome c, but different from cytochrome c-551 from Pseudomonas aeruginosa. These results provide additional support for a previously proposed correlation between the chirality of attachment of the axial methionine and the electronic wave functions in oxidized cytochromes of the c type. Comparison of mammalian cytochrome c, cytochrome c-551 and cytochrome c-552 indicates that the chirality of the axially bound methionine is not linked with the evolutionary increase of the polypeptide chain length.