Myoglobin heme -reconstituted

Figure 12. MCD spectra of the pyridine hemochromogens (ferrous bis-pyridine adducts) of alkaline-denatured Spirographis heme-reconstituted myoglobin (-----) and myeloperoxidase (-----) (both in A) and of iron oc-...

M. Sono, A.M. Bracete, A.M. Huff, M. Ikeda-Saito, and J.H. Dawson, Evidence that a formyl-substituted iron porphyrin is the prosthetic group of myeloperoxidase magnetic circular dichroism similarity of the peroxidase to Spirographis heme-reconstituted myoglobin, Proc. Natl. Acad. Sci. USA 88 11148 (1991). 

Hamachi I,Tsukiji S, Shinkai S, Oishi S. Direct observation of the ferric-porphyrin cation radical as an intermediate in the phototriggered oxidation of ferric- to ferryl-heme tethered to Ru(bpy)3 in reconstituted myoglobin. J Am Chem Soc 1999 121 5500-6. 

One of the variables in the structures of the porphyrins present in heme proteins is the presence or absence of vinyl substituents on the periphery of the macrocycle. For example, b hemes have vinyl substituents whereas c hemes do not. Because of the sensitivity of such vinyl substituents during synthetic transformations, it has often been desirable to use octa-alkyl porphyrins in model studies of the spectroscopic properties of heme systems. The development of improved methods for the preparation of octa-alkyl porphyrins has likewise increased the availability of such porphyrins for model studies (20, 21). To assess the effect that replacement of the two vinyl substituents in protoporphyrin IX with alkyl (ethyl) groups has on the MCD properties of the heme system, an extensive and systematic study of the MCD properties of mesoheme IX-reconstituted myoglobin and horseradish peroxidase in comparison with the spectra of the native protoheme-bound proteins has been carried out (22). The structures of these two porphyrins are shown in Figure 3. 

Several MCD comparisons have been reported involving intact (i.e., nondenatured) myeloperoxidase and heme s-reconstituted myoglobin (43). The heme s-reconstituted myoglobin has been investigated as a model for myeloperoxidase because it has a histidine proximal ligand as... 

Scheme 1 Reconstitution of myoglobin with an artificially created heme...

Figure 3-1. The reconstitution of apo-myoglobin with functionalized heme units.

Figure 8. Infrared difference spectra of carbonyl sperm whale myoglobins reconstituted from different hemes vs. metmyoglobin in phosphate buffer, pH 6 4, Top protoheme. Middle deu-teroheme. Bottom mesoheme.

Fluorinated analogues of these and related porphyrins were later used to reconstitute myoglobins that contain fluorinated hemes. These were used for NMR studies of the effects of distortion of the porphyrin 7t-system by the chemical properties of the peripheral side chains. A similar strategy involving the oxidative coupling of similar dipyrromethenes and monofluorodipyrromethenes produced 6-monofluorinated porphyrins. Reconstituted myoglobins and iron complexes prepared from these were studied. ... 

CD can be used to monitor the phenomenon of heme isomerism in heme proteins. " The isomerism, discovered by LaMar and co-workers, results from the presence of two forms of myoglobin which differ by 180° in the heme orientation about the a, y-methine carbon axis. The two isomers are present in a 9 1 ratio at equilibrium in carbonmonoxymyo-globin (MbCO) but are in a 1 1 ratio in a sample freshly reconstituted from apomyoglobin and heme. Freshly reconstituted MbCO has a Soret CD band with only about half the amplitude of the native form, " suggesting that the minor isomer (at equilibrium) has only a weak Soret CD spectrum. The major form has a strong positive Soret CD band, whereas the minor form has a weakly negative Soret CD band. These results and the CD of the heme undecapeptide from cytochrome indicate that the origin of the Soret CD band must be more complex than proposed by Hsu and Woody, ... 

These studies prompted a reexamination of the stimulatoiy effects of apo cyt 65 in a reconstituted system with P450 3A4 and 17A1 [133], The reexamination concluded that far less heme transfer occurred than could be accounted for by the stimulatoiy effects of apo cyt by Furthermore, apo myoglobin did not inhibit the stimula-toiy effects of apo cyt by The reexamination did not include investigation of the effects of redox inactive cyt b which had been reconstituted with a Zn-substituted protoporphyrin IX. Nor did it investigate whether cyt b mutants that were unable to bind heme were still stimulatoiy. 

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