Reengineering natural design by rational design and in vivo library selection: the HLH subdomain in bHLHZ proteins is a unique requirement for DNA-binding function.

To explore the role of the HLH subdomain in bHLHZ proteins, we designed sets of minimalist proteins based on bHLHZ protein Max, bHLH/PAS protein Arnt and bZIP protein C/EBP. In the first, the Max bHLH and C/EBP leucine zipper were fused such that the leucine heptad repeats were not in register; therefore, the protein dimerization interface was disrupted. Max1bHLH-C/EBP showed little ability to activate transcription from the E-box (5'-CACGTG) in the yeast one-hybrid assay, and no E-box binding by quantitative fluorescence anisotropy.

The ferrous verdoheme-heme oxygenase complex is six-coordinate and low-spin.

A biosynthetic and enzymatic method was developed for the preparation of 13C-labeled verdoheme, which permits the 13C NMR spectroscopic characterization of this elusive intermediate in the heme oxidation path catalyzed by the enzyme heme oxygenase. The 13C NMR data indicate that the ferrous verdoheme complex of Neisseria meningitides heme oxygenase is hexacoordinate and diamagnetic, with a proximal histidine and likely a distal hydroxide as axial ligands. The coordination number and spin state of the ferrous verdoheme-heme oxygenase complex is in stark contrast to the pentacoordinate and paramagnetic nature of the heme-heme oxygenase complex and heme centers in general.

Reduction of the ferrous alpha-verdoheme-cytochrome b5 complex.

The ferrous alpha-verdoheme-cytochrome b(5) complex, [Fe(II)(verdoheme)](+), has been prepared and characterized spectroscopically. Anaerobic addition of excess sodium dithionite to [Fe(II)(verdoheme)](+) at pH 10 produces a one-electron-reduced species with spectroscopic characteristics that suggest a ferrous hexacoordinated verdoheme pineutral radical best formulated as a [Fe(II)(verdoheme*)] --> [Fe(I)(verdoheme)] resonance hybrid. At lower pH values (7.

Coupled oxidation vs heme oxygenation: insights from axial ligand mutants of mitochondrial cytochrome b5.

Mutation of His-39, one of the axial ligands in rat outer mitochondrial membrane cytochrome b(5) (OM cyt b(5)), to Val produces a mutant (H39V) capable of carrying out the oxidation of heme to biliverdin when incubated with hydrazine and O(2). The reaction proceeds via the formation of an oxyferrous complex (Fe(II)(-)O(2)) that is reduced by hydrazine to a ferric hydroperoxide (Fe(III)(-)OOH) species. The latter adds a hydroxyl group to the porphyrin to form meso-hydroxyheme.