Regulatory factors affecting ferrochelatase activity were studied and an attempt was made to determine the role of ferrochelatase in the regulation of heme biosynthesis. Ferrochelatase was found to have a Km value of 0.105 mM for the porphyrin substrates, proto and mesoporphyrin IX and a Km value of 8.30 x 10⁻³ mM for ferrous ion, its metal substrate. The Vmax values for proto and mesoporphyrin IX were 12.05 and 28.57 units/mg, respectively, and that of ferrous ion was 2.89-units/mg. Ferrochelatase exhibited feedback product inhibition by hemin in concentrations between 1 and 10 uM and stimulation of ferrochelatase activity by hemin at concentrations above 20 uM. Concentrations of ferrous ion exceeding 0.25 mM were found to inhibit ferrochelatase activity, indicating that the enzyme is subject to substrate inhibition. The iodoacetamide sensitive binding site of ferrochelatase was determined to be on the inside of the inner mitochondrial membrane in contact with the matrix. Ferrochelatase activity was found to be sensitive to its membrane environment, in particular it was dependent on the hydrophobic portion of the phospholipids for activity rather than their hydrophilic head groups. This was demonstrated in experiments in which the lipids were removed from submitochondrial particles or detergent-solubilized preparations of rat liver mitochondria by acetone extraction, and ferrochelatase activity reconstituted by the addition of lipids. Reactivation was found to be a function of the unsaturation of the acyl chain of either the fatty acid or phospholipid. Cholesterol was found to increase activity below 2 8°C and to decrease activity above 45°C. Discontinuities were seen in Arrhenius plots of ferrochelatase at 37°C for submitochondrial particles and at 28.5°C for detergentT-solubilized preparations. Ferrochelatase was shown to have an absolute requirement for calcium ions but this was not a requirement of the ferrochelatase protein, rather, it was mediated through some effect of calcium on the membrane. Ferrochelatase was observed to have an absolute requirement for ferrous ion as metal substrate and to be able to utilize ferric ion only in the presence of electron donors such as NADH, NADPH, succinate, ⍺-glycerol phosphate or choline chloride. The recovery of electrons from the donors for iron reduction was dependent upon the presence of their respective dehydrogenases and was independent of respiration or energy processes. In addition to providing reducing equivalents for iron reduction, the electron donors also stimulated ferrochelatase activity. Mitochondria from anaerobically grown Saccharomyces cerevisiae were found to have high ferrochelatase activity but to have no iron reduction activity, whereas mitochondria from aerobically grown S. cerevisiae possessed both high ferrochelatase and iron reducing abilities. The appearance of iron reduction ability during respiratory adaptation of yeast was found to correlate closely with the appearance of respiratory enzymes and to be one of the first activities detected after the onset of aerofoiosis. Conventional techniques were insufficient to separate the ferrochelatas and iron reductase activities, although an assay based on the reduction of PMS by ferrous ion was used to quantitate iron reduction activity independently. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

by Kwong-kwok Wong. / Thesis (M.Ph.)--Chinese University of Hong Kong, 1987. / Bibliography: leaves 158-164.

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Thesis (Ph.D.)--University of Delaware, 2008. / Principal faculty advisor: Roberta Colman, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

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Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 176-207). Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 176-207). Also available in print.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 196-212).

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 190-207) Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 190-207) Also available online.