Magnetic circular dichroism analysis of the IHP effect on spin equilibria in human ferric hemoglobins.

Magnetic circular dichroism (MCD) spectroscopy has been to explore the connection between optical spectra and the high spin population of several hemoglobins under various conditions. It is found that the effectiveness of IHP in inducing spectral changes can be markedly affected by solvent. For example, the IHP-induced spectral changes in the visible region for nitritomethemoglobin-A in mixed buffer solvent systems (glycerol or polyethylene glycol (PEG), mw 190-210) are more than double those observed in aqueous buffers. We estimate that IHP induces a mix of R/T forms in bis-tris phosphate buffers, for NO-2metHb that is only about 50%. While PEG and glycerol both lead to enhanced IHP-induced spectral differences, they behave differently in two aspects. PEG shifts the visible MCD and absorption spectra of F-metHb-A, supposedly already biased towards the T form by ligand, in the same direction that IHP does. PEG also maximizes the spin state changes with IHP for three R from hemoglobins and N-3metHb-A, and so appears to stabilize the T form in all cases. Glycerol does not. In addition, the apparent binding constant for NO-2 to H2OmetHb-A differs between these two solvents. Comparison of the data from several hemoglobins leads to the conclusion that the changes in spin state distributions induced by IHP correlate well with quaternary structure for a given hemoglobin. An analogous correlation amongst various proteins between initial spin state distribution (IHP) absent) and quaternary structure is not found.

Magnetic circular dichroism of ferrous carbonyl adducts of cytochromes P-450 and P-420 and their synthetic models: further evidence for mercaptide as the fifth ligand to iron.

Absorption and magnetic circular dichroism (MCD) spectra have been obtained for the ferrous carbonyl adducts of cytochromes P-450 and P-420 as well as synthetic model systems. Ferrous porphyrins with sodium methyl mercaptide and CO in benzene give MCD and absorption spectra which are almost identical to those of the natural enzyme, indicating that in P-450 a mercaptide serves as the fifth ligand in the ferrous carbonyl adduct. MCD spectra of models with either propyl mercaptan or N-methylimidazole as the axial ligand are identical with that of P-420. Thus, no unambiguous assignment of the axial ligand can be made in this case. The infrared stretching frequencies of ferrous porphyrin carbonyl complexes and the absorption spectrum of the CO adduct of Na[Fe1(meso-tetraphenylporphyrin dianion)] are consistent with the concept that in P-450 considerable electron density is transferred to the iron by the mercaptide ligand.

Magnetic circular dichroism studies XXXV. A comparison of cytochromes P-450 and P-448.

Cytochromes P-450 and P-448 in microsomal suspensions have been shown to be spectrally distinct by magnetic circular dichroism spectroscopy. Furthermore, this technique can be used to measure induction of these two cytochromes by phenobarbital and 3-methyl-cholanthrene. Magnetic circular dichroism spectroscopy is thus at least as useful as difference spectroscopy for the investigation of P-450 and P-448 and more informative because the presence of cytochrome b(5) and hemoglobin can be detected concurrently. We have also shown that the molar magnetic ellipticity for reduced + CO treated cytochrome P-450 of Pseudomonas grown on camphor is of similar value to that of reduced + CO treated microsomal P-450 and P-448.

Magnetic circular dichroism studies. XXV. A preliminary investigation of microsomal cytochromes.

The application of magnetic circular dichroism as an optical probe for simultaneous identification and determination of at least two microsomal cytochromes is demonstrated. The assignments of the bands in the spectra of microsomal suspensions are made from the spectra of soluble preparations of cytochrome P-450 obtained from Pseudomonas putida and of cytochrome b(5) obtained from rat livers.