Ferryl myoglobin

Dee, G., Rice-Evans, C., Obeyesekera, S., Meraji, S., Jacobs, M. and Bruckdorfer, K.R. (1991). The modulation of ferryl myoglobin formation and its oxidative effects on low density liproprotein by nitric oxide. FEBS Lett. 294, 38-42. 

In order to understand the potential for haem proteins to mediate the oxidative modification of LDLs, the interaction between ruptured erythrocytes (Paganga et al., 1992) and ruptured myocytes (Bourne etal., 1994) with LDL has been explored. Previous studies from this group have demonstrated that ferryl myoglobin radicals and ruptured cardiac myocytes, which generate ferryl myoglobin species on activation (Turner et al., 1990,... 

As described above, efficient peroxidase catalysis requires rapid reaction of the enzyme with H2O2 coupled with the formation of a discrete compound I species. As initially observed by George and Irvine in 1952 (177), the reaction of metMb with H2O2 is much slower than the corresponding reaction of peroxidases. The myoglobin derivative produced by this reaction was referred to by these authors as ferryl myoglobin... 

Some haem proteins undergo a peroxidase cycle like that in Fig. 2, but very slowly, e.g. myoglobin converting to ferryl myoglobin. In this case the reaction is presumed not to have a physiological role. Indeed, it can have deleterious consequences (see section 5). 

Fig. 8. MCD spectra of ferryl iron. Low-temperature (50 or 100K) MCD spectra of ferryl iron in different proteins HRPCII, horse-radish peroxidase compound II HRPCX, horse-radish peroxidase compound X YCCP, yeast cytochrome c peroxidase compound I PsCCP, compound I of the dihaem cytochrome c peroxidase from Pseudomonas aeruginosa-, Mb pH 3.5, ferryl myoglobin formed at pH 3.5 MbpD9.0, the same compound found at pD9.0. Note the similarity of all the spectra with the exception of the alkaline form of ferryl myoglobin. Reprinted with permission from Cheesman, M.R., Greenwood, C. and Thomson, A.J. (1991) Adv. Inorg. Chem. 36, 201-255.

MCD spectra perhaps provide the best fingerprint for the existence of an FeIV=0 structure. Fig. 8 shows that there is a great similarity between the spectra of horseradish peroxidase compound II, horseradish peroxidase compound X, cytochrome c peroxidase compound I, Pseudomonas aeruginosa peroxidase compound I and ferryl myoglobin at acid pH. Similar features are seen in the spectra of catalase [170] and myoglobin [171] compound II. 

The reasons for the difference between the spectra of ferryl myoglobin at acid and alkaline pH are not clear. However, it has been suggested that deprotonation of the proximal histidine ligand at alkaline pH may be responsible [175,176], Furthermore a third form of ferryl iron was detected in varying amounts in preparations of horseradish peroxidase compound II, ferryl myoglobin and cytochrome c peroxidase compound I [162], To account for these spectra it was proposed that the iron-histidine bond was broken, leaving a five-coordinate ferryl haem. 

Mossbauer spectra has been extensively used to probe the structure of the iron nucleus in biological FeIV=0 compounds. These include horseradish peroxidase compoundl[134,180,181], horseradish peroxidase compound II [182,183], horseradish peroxidase compound X [181], Japanese-radish peroxidase compounds I and II [184], chloroperoxidase compound I [185], cytochrome c peroxidase compound I [186] and ferryl myoglobin [183]. Examples of Mossbauer spectra attributed to non-porphyrin-bound FeIV are only available from synthetic model compounds. These include compounds with [130] and without [4-8] an FeIV=0 bond. 

With ferryl myoglobin, in contrast to peroxidases, the reactions of the protein free radicals and that of the ferryl haem can be considered as uncoupled from each other. The protein has not been designed to form a cation radical for a specific reaction therefore not only is more than one cation free radical generated, but there is no control over their subsequent reactions. A similar situation can be observed in cytochrome c peroxidase mutants that have lost tryptophan-191. A different amino-acid free radical is still formed that is less stable. Indeed, even in the presence of tryptophan-191, small amounts of other free radicals are formed [237] this is further evidence that even in enzymes it is difficult to exclusively control free radical reactions. 

Ferryl myoglobin species (either the FeIV=0 itself or the protein free radicals) are capable of catalysing lipid peroxidation in model membranes [238], erythrocytes [239] and low-density lipoproteins [240]. They can also oxidise phenols, styrene, 3-carotene and ascorbate [211], At high peroxide levels, protein cross-linking is observed, followed by iron release which can result in Fenton chemistry in vitro. However, it is difficult to see how the peroxide haem ratio can ever be high enough in vivo to initiate these reactions. 

Ferryl myoglobin has been detected indirectly in vivo by the addition of sulfide to generate sulfmyoglobin. This derivative can then be detected... 

Fig. 10. EPR spectra of the effect of (upper panel) desferrioxamine and (lower panel) A -methyl-V-acyl hydroxamate on ferryl myoglobin species.

Ferryl myoglobin can be identified spectrophotometrically, although it is not possible to differentiate between the radical and nonradical state using this technique. Whereas metmyoglobin shows characteristic peaks at 535, 585 and 630 nm, ferrylmyoglobin has peaks... 

Fig. 4.5. ESR spectrum of the ferryl myoglobin radical (Turner et al., 1990). ESR spectrum observed on reaction of (a) metmyoglobin (225 //M) and (b) oxymyoglobin with 250 / M hydrogen peroxide in the presence of 25 mM DMPO at pH 7.4 under normoxic...

These equations are applicable to ferryl myoglobin radical production in chemical systems, but the results are less clear when applied to cellular systems such as cardiac myocytes. A better spectroscopic approach here is to observe the shifts in the Soret region to detect qualitatively the presence of ferryl myoglobin (Turner et al., 1991). Fig. 4.6 shows the comparison between the visible spectra of metmyoglobin... 

Fig. 4.6. Spectroscopic identification of ferryl myoglobin. The activation of metmyoglo-bin to ferryl myoglobin by hydrogen peroxide (x 1.25 molar excess) the development of the characteristic peaks of ferryl myoglobin as a function of time (minutes) (a) 0, (b) 0.5, (c) 2.5, (d) 4.5, (e) 6.5 and (0 8-5. The development of the characteristic peaks for ferryl myoglobin around 550 and 580 nm is shown, with a decrease in the shoulder at 630 nm, characteristic of metmyoglobin, and the peak at 515 nm.

If chemical studies are set up to investigate the formation and reactions of ferryl myoglobin, the metmyoglobin and oxymyoglobin must be purified. 

Sitter AJ, Reczek CM, Temer J (1985) Observation of the Fe -0 stretching vibration of ferryl myoglobin by resonance Raman spectroscopy. Biochimica Et Biophysica Acta (BBA) Protein Struct Mol Enzymol 828 229-235... 

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