Richard J. Kassner

Heme formation from Fe(II) and porphyrin in the absence of ferrochelatase activity

Abstract Characteristics of the reaction of iron with porphyrin have been studied in aqueous solution in the absence of cellular material according to an established set of conditions used for the assay of cell preparations for ferrochelatase activity. Chelation of iron by porphyrin occurred only with Fe(II) and under anaerobic conditions. The reaction was activated by GSH when Fe(III) was added to the reaction mixture and inhibited by GSH when iron was present in its divalent state. The rate of heme formation was dependent on the nature of the porphyrin side-chains and completely inhibited by esterification of the carboxylic acid side-chains. pH optima of 8.25 and 9.5 were demonstrated for hemato- and mesoheme formation, respectively. The reaction was strongly inhibited by the detergent Tween 80 at concentrations as low as 0.05%. In addition, the results indicated that the heme formed with the dicarboxylic acid porphyrins was equivalent to or greater than that reported for the reaction in the presence of ferrochelatase preparations. A comparison of these results with reported properties of the reaction catalyzed by cell-free preparations is made and criteria suggested for the future assignment of enzyme activity to cell extracts.

Electrochemistry and ESR spectroscopy of hemin and hemeoctapeptide from equine cytochrome-c with several ligands

The cyclic voltammograms and ESR spectra are reported for a series of ligands coordinated to hemin and the ferric hemeoctapeptide (H8PT). The ligands consist of small anions as well as five membered and six membered aromatic and nonaromatic heterocycles with nitrogenous and oxygenous donor atoms. The electrochemistry of the heme-ligand complexes is studied in DMF solution. The axial imidazole coordination from the histidine in H8PT has the effect of attenuating the potential shift caused by the ligand occupying the trans axial position.

Spectroscopic properties of ferrous heme complexes of sterically hindered ligands

Mesoheme IX complexes of sterically hindered ligands 2-methylimidazole, tert-butylamine and 2-methylpyridine in aqueous glycerol solutions are characterized by broad visible absorption spectra at ambient temperature exhibiting close similarities to high-spin ferrous hemeproteins. Spectrophotometric titrations of mesoheme IX with these ligands indicate well-defined equilibria for 2-methylimidazole and tert-butylamine corresponding to the formation of penta-coordinate strong-field ligand complexes. Variable temperature spectra of these complexes from ambient to 77 degrees K exhibit a change to hemochrome spectra characteristic of the low-spin unhindered ligand complexes. Corresponding changes in the visible spectra are not observed for the high-spin hemeproteins deoxymyoglobin, horse-radish peroxidase and cytochrome ć. The appropriate utilization of these hindered ligand heme complexes as model systems for high-spin ferrous hemeproteins has been discussed.

Binding of ethyl isocyanide to cytochrome c′ from Chromatium vinosum

Abstract Alkyl isocyanides were investigated as potential ligands for the cytochromes c ′. Spectroscopic evidence is presented which demonstrates the binding of ethyl isocyanide to cytochrome c ′ from Chromatium vinosum . Chromatium cytochrome c ′ was shown to bind one molecule of ethyl isocyanide with an equilibrium constant of 3.29·10 3 at pH 7.0 and 25°C. This new finding indicates that the binding site of Chromatium cytochrome c ′ may be significantly more accessible than that of Rhodospirillum rubrum cytochrome c ′ and further provides an opportunity to examine the structure of the heme environments of the cytochromes c ′ through a study of the binding of alkyl isocyanides to these proteins.

Spectroscopic properties of low-spin ferrous heme complexes and hemeproteins at 77°K

Abstract The low temperature optical spectra in the region of the Q 00 (α-band) and Q 01 (β-band) transitions of model heme complexes for b - and c-type cytochromes were measured and the results discussed in terms of the similarities and differences to the spectra of horse heart cytochrome c and other hemeproteins. Comparisons of the resolved vibronic components of the Q 01 and β′ bands were made to the recent resonance Raman spectra of hemeproteins. Tentative assignment of the β′ band to Q 02 type transitions has been proposed.

The effect of temperature on the spectroscopic properties of the heme c octapeptide from cytochrome c

The effect of temperature on the optical properties of the acetylated heme c octapeptide from cytochrome c was examined. At above ambient temperatures the observed optical spectrum with maxima at 549 and 424 nm was characteristic of high-spin ferrous hemeproteins. At below ambient temperatures the optical spectrum became characteristic of low-spin ferrous hemeproteins with maxima at 547, 518, and 410 nm. A thermodynamic characterization of this two component system yielded a deltaHO of -10.1 +/- 0.7 kcal/mol and a delta S0 of-37.6 +/- 2.5 e.u. for the temperature dependent process. Discussion of the spectroscopic and thermodynamic parameters was presented in terms of the consistent magnetic and structural properties of heme complexes.