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. Max1bHLH-C/EBP's activity was significantly improved after library selection (three amino acids randomized between HLH and leucine zipper), despite the Max bHLH and C/EBP zipper still being out of register: a representative mutant gave a high nanomolar K(d) value for E-box binding. Thus, selection proved to be a powerful tool for salvaging the flawed Max1bHLH-C/EBP, although the out-of-register mutants still did not achieve the strong DNA-binding affinities displayed by their in-register counterparts. ArntbHLH-C/EBP hybrids further demonstrated the importance of maintaining register, as out-of-register mutants showed no E-box-responsive activity, whereas the in-register hybrid showed moderate activity. In another design, we eliminated the HLH altogether and fused the Max basic region to the C/EBP zipper to generate bZIP-like hybrids. Despite numerous designs and selections, these hybrids possessed no E-box-responsive activity. Finally, we tested the importance of the loop sequence in MaxbHLHZ by fluorescence and circular dichroism. In one mutant, the loop was shortened by two residues; in the other, the Lys57:DNA-backbone interaction was abolished by mutation to Gly57. Both showed markedly decreased E-box-binding relative to MaxbHLHZ. Our results suggest that, in contrast to the more rigid bZIP, the HLH is capable of significant conformational adaptation to enable gene-regulatory function and is required for protein dimerization and positioning the basic region for DNA recognition.

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 (7.0 and 8.0) the one-electron-reduced species is shown to disproportionate to produce the resting state [Fe(II)(verdoheme)](+) complex and the two-electron-reduced [Fe(II)(verdoheme:)](-) anion. The latter might also be formulated as a resonance hybrid [Fe(I)(verdoheme*)](-) --> [Fe(II)(verdoheme:)](-). The disproportionation reaction becomes very slow as the pH is raised above 9.0. Exposure of the one-electron- or two-electron-reduced verdoheme complexes of cytochrome b(5) to O(2) results in rapid and quantitative reoxidation to the resting state [Fe(II)(verdoheme)](+) complex.

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. The observation that catalase does not inhibit the oxidation of the heme in the H39V mutant is consistent with the formation of a coordinated hydroperoxide (Fe(III)(-)OOH), which in heme oxygenase is the precursor of meso-hydroxyheme. By comparison, mutation of His-63, the other axial ligand in OM cyt b(5), to Val results in a mutant (H63V) capable of oxidizing heme to verdoheme in the absence of catalase. However, the oxidation of heme by H63V is completely inhibited by catalase. Furthermore, whereas the incubation of Fe(III)(-)H63V with H(2)O(2) leads to the nonspecific degradation of heme, the incubation of Fe(II)(-)H63V with H(2)O(2) results in the formation of meso-hydroxyheme, which upon exposure to O(2) is rapidly converted to verdoheme. These findings revealed that although meso-hydroxyheme is formed during the degradation of heme by the enzyme heme oxygenase or by the process of coupled oxidation of model hemes and hemoproteins not involved in heme catabolism, the corresponding mechanisms by which meso-hydroxyheme is generated are different. In the coupled oxidation process O(2) is reduced to noncoordinated H(2)O(2), which reacts with Fe(II)-heme to form meso-hydroxyheme. In the heme oxygenation reaction a coordinated O(2) molecule (Fe(II)(-)O(2)) is reduced to a coordinated peroxide molecule (Fe(III)(-)OOH), which oxidizes heme to meso-hydroxyheme.