Magnetic circular dichroism spectroscopic characterization of the L358P mutant of cytochrome P450-CAM, Geobacillus stearothermophilus nitric oxide synthase, the mammalian transcription factor neuronal PAS protein 2 and heme protein maquettes.

摘要

Magnetic circular dichroism spectroscopy (MCD) has been used to characterize the L358P mutant of cytochrome P450-CAM and a recombinant nitric oxide synthase (NOS) isolated from the bacterium Geobacillus stearothermophilus (gsNOS). Spectral comparisons of ligand complexes of L358P P450-CAM and gsNOS with analogous wild-type P450-CAM and mammalian NOS complexes indicate overall spectroscopic similarities between L358P and wild-type P450-CAM and between gsNOS and mammalian NOS. Detailed spectral comparisons of gsNOS and mammalian NOS, particularly of the key oxyferrous intermediate, indicate subtle structural differences. Additionally, formation of the stable ferric-NO product complex was observed spectroscopically upon addition of O₂ to deoxyferrous gsNOS at -35°C or -50°C in the presence of excess NHA and BH₄.;MCD has also been used to investigate the heme-iron axial ligation of the PAS heme domains of the mammalian transcription factor neuronal PAS protein2 (NPAS2) and of synthetic heme protein maquettes. MCD spectral data suggest bis-histidine ligation in the ferric and ferrous states of the PAS domains, with CO-binding displacing one of the histidine ligands. Comparisons between the MCD data presented in this work and other spectroscopic studies indicate that the heme environment of the NPAS2 PAS domains may be flexible and may use alternate amino acids as axial ligands. Heme protein maquettes were designed to bind a five-coordinate mono-(1-methyl-histidine)-ligated heme, bis-histidine-ligated heme b, and a bis-histidine-ligated mimic of heme a. The MCD data indicate that the mono-(1-methyl-histidine)-ligated heme is a spectroscopic model of ferrous myoglobin and that the heme a mimic is a spectroscopic model for the bis-histidine-ligated heme a found in cytochrome c oxidase.