5-Aminolevulinate synthase (ALAS) is a mitochondrial enzyme that catalyzes the first step of the heme biosynthetic pathway. The mitochondrial import, as well as the synthesis, of the nonspecific isoform of ALAS (ALAS1) is regulated by heme through a feedback mechanism. A short amino acid sequence, the heme regulatory motif (HRM), is known to be involved in the regulatory function of heme. To determine the role of the HRM in the heme-regulated transport of the nonspecific and erythroid forms of ALAS in vivo, we constructed a series of mutants of rat ALAS1, in which the cysteine residues in the three putative HRMs in the N-terminal region of the enzyme were converted to serine ones by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in quail QT6 fibroblasts through transient transfection, and the mitochondrial import of these enzymes was examined in the presence of hemin. Hemin inhibited the mitochondrial import of wild-type ALAS1, but this inhibition was reversed on the mutation of all three HRMs in the enzyme, indicating that the HRMs are essential for the heme-mediated inhibition of ALAS1 transport in the cell. By contrast, exogenous hemin did not affect the mitochondrial import of the erythroid-specific ALAS isoform (ALAS2) under the same experimental conditions. These results may reflect the difference in the physiological functions of the two ALAS isoforms.

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In this kinetic and thermodynamic study, the reversible outer-sphere electron-transfer reactions between a series of Ru(NH(3))(5)L(3+/2+) complexes (L = etpy, py, lut) (etpy = 4-ethylpyridine; py = pyridine; lut = 3,5-lutidine) and cytochrome c were investigated as a function of ionic strength, buffer, pH, temperature, and pressure. Due to the low driving forces of these systems, it was possible to study all the reactions in both redox directions. The observed rate constants for various L are correlated on the basis of the ability of ligands on the ruthenium complex to penetrate the heme groove on cytochrome c. The measurements as a function of pressure enabled the construction of volume profiles for all investigated systems. The activation volumes for all of these processes are very similar: between -14.9 and -17.8 cm(3) mol(-)(1) for the reduction and between +14.7 and +17.8 cm(3) mol(-)(1) for the oxidation of the protein by Ru(NH(3))(5)L(2+/)(3+), respectively. The overall reaction volume varies between 27 and 35 cm(3) mol(-)(1), from which it follows that the transition state lies exactly halfway between reactant and product states on a volume basis in all cases. There is good agreement throughout between kinetic and thermodynamic data.

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Several ruthenium ammine complexes were used to modify horse-heart cytochrome c at histidine-33, creating a series of (NH 3 ) 4 (L)Ru-Cyt c derivatives (L ) H 2 O/OH -, ammonia, 4-ethylpyridine, 3,5-lutidine, pyridine, isonicotinamide, N-methylpyrazinium) with a wide range of driving forces for Fe-to-Ru electron transfer (-∆G°) -0.125 to +0.46 eV). Electron-transfer rates and activation parameters were measured by pulse radiolysis using azide or carbonate radicals. The driving-force dependence of electron-transfer rates between redox centers of the same charge types obeys Marcus-Hush theory. The activationless rate limit for all of the ruthenium derivatives except the N-methylpyrazinium complex is 3.9 × 10 5 s -1 . Thermodynamic parameters obtained from nonisothermal differential pulse voltammetry show that the electron-transfer reactions are entropy-driven. The thermodynamic and kinetic effects of phosphate ion binding to the ruthenium center are examined. The rate of intramolecular electron transfer in (NH 3 ) 4 (isn)Ru III -Cyt c II decreases at high pH, with a midpoint at pH 9.1.