Heme proteins and oxidation in fresh and processed meats - Oxidation in Foods and Beverages and Antioxidant Applications - page 89

Ser(F7) and His(FG3)in Mbs interact with the heme-7-propionate while
Leu(F7) and Leu(FG3) in Hb do not so that the porphyrin group is more
`anchored' in Mbs. Evidence for this can be seen when examining Mb mutants
in which the native residues at F7 and FG3 were replaced with residues identical
or similar to those found in Hbs at the same site. For example, the Ser(F7)Leu
Mb mutant released its hemin moiety 20-fold faster compared to WT Mb
(Smerdon et al., 1993). The His(FG3)Val mutant released its hemin moiety 23-
fold more rapidly compared to WT Mb (Hargrove et al., 1996).
It should be noted that the tendency of Hb tetramers to dissociate to
monomers and dimers accelerates hemin loss and Hb oxidation (Griffon et al.,
1998; Hargrove et al., 1997). Enhanced ionic strength and dilution of Hb
promotes dissociation of Hb tetramers to subunits (Antoni and Brunoni, 1971;
Manning et al., 1998). There have been reports that decreasing pH (in the pH
range of 7 to 5) enhances Hb subunit formation but this may be due to effects
from using acetate buffer. An Hb mutant (rHb 0.1) with decreased ability to
form subunits was a weaker promoter of lipid oxidation compared to wild type
Hb (Grunwald and Richards, 2006a).
The reaction of nitrite with oxyMb resulted in the formation of a ferryl Mb
radical while reaction of nitrite with oxyHb did not (Keszler et al., 2006).
Formation of the Mb radical was inhibited by catalase indicating involvement of
hydrogen peroxide in Mb radical formation. The ferryl heme protein radical is
capable of abstracting hydrogen atoms from polyunsaturated fatty acids which
can initiate lipid oxidation (reaction 11).
4.6.3 Role of released hemin compared to other oxidative forms of Hb and
Mb
The challenge in understanding the pathway by which heme proteins promote
lipid oxidation is that heme protein autooxidation, ferryl radical formation,
hemin release, heme protein crosslinking, hemichrome formation, and iron
release can all occur in a very short time sequence (and simultaneously) so that
the most relevant step related to lipid oxidation is obscured. However, amino
acid substitutions of native Hb and Mb can be used to manipulate various
properties of the heme proteins. For example, the ability of the hemin porphyrin
to remain attached to the globin can be varied 975-fold by comparing wild-type
sperm whale Mb with V68T and H97A (Hargrove et al., 1996). Substitution of
valine at site E11 with threonine increases hemin affinity 25-fold. Thr(E11) will
hydrogen bond with liganded water increasing hemin affinity while the native
valine cannot hydrogen bond with liganded water (Fig. 4.5). Substituting
histidine at site FG3 with alanine decreases hemin affinity 39-fold. The smaller
alanine at FG3 allows water to rapidly enter into the heme crevice which
hydrates the proximal histidine lowering hemin affinity; the native histidine at
FG3 sterically blocks water from the proximal side of the heme crevice and
chemically bonds with the heme-7-propionate (Fig. 4.6). The different mutants
are separately added to washed fish muscle at post-mortem pH to assess the