STRESS RESPONSES - Cyanobacteria - page 691

H-bond network involving Trp122, His123 and Arg119, the haem propionate group and Trp106
plays a key role on the formation of Trp • radical.
The existence of a covalent bond between Trp122, Tyr249 and Met275 and the formation of a
novel covalent adduct has been demonstrated by the mass spectrometric analysis of the wild-type,
recombinant and the corresponding variants (Trp122, Tyr249 and Met 275) of KatG enzymes from
Synechocystis sp. strain PCC 6803 (Jakopitsch et al ., 2003d). As already mentioned the exchange of
Trp122 with Phe or Ala (Regelsberger et al ., 2000) and Tyr249 with Phe (Jakopitsch et al ., 2003c) resulted
in a complete loss of catalase activity but not peroxidase activity. The cross-linking of Trp122 and
Tyr249 is thus important in catalase activity as variants of Met275 still allowed the covalent bond
formation between Trp122 and Tyr249 (Jakpoitsch et al ., 2003d). The variants of Met275 showed a
reduced catalase activity whereas the peroxidase activity is normal (Donald et al ., 2003). Further,
the novel covalent adduct (of Trp122, Tyr249 and Met275) appears to be hydrogen bonded to a
neighbouring Arg439 of Synechosystis KatG enzyme. In the KatG of S . elongatus PCC 7942 Arg409
is at H-bonding distance to both Tyr218 and Met244. A lower turnover number of catalase activity
has been observed in case of mutant enzymes of KatG (Tyr249Phe, Met275Ile, Arg439Ala and
Arg439Asn) of Synechocystis sp. strain PCC 6803 but the peroxidase activity remained unaffected.
The typical KatG covalent adduct remained intact in the two Arg439 variants as in case of wild-
type. In the Met275Ile variant the covalent link existed only between Tyr249 and Trp122 whereas in
case of Tyr249Phe variant the link is absent. The radical species formed in the variants Tyr249Phe
and Arg439Ala/Asn during their interaction with peroxoacetic acid was similar to wild-type and
they are oxoferrylporphyrin radical, tryptophanyl radical and the tyrosyl radical. The Met275Ile
variant did not form the Trp • species probably due to change in the haem distal site. The sequence of
intermediates has been noted to be por •+ , Trp • (or Trp •+ ) and Tyr • (Jakopitsch et al ., 2004). The reaction
of KatG of Synechocystis sp. strain PCC 6803 with isoniazid resulted in the identifi cation of the fi rst
compound I unequivocally as [Fe(IV)=O Por •+ ]. Subsequent to this, it was possible to identify two
protein-based radicals Trp106 • and a Tyr • formed by intramolecular electron transfer. Further, the
reaction of isoniazid with KatG enzyme seemed to be very close to the haem distal side that could
readily react with [Fe(IV)=O Por •+ ] species. However, the KatG from M . tuberculosis differed in its
reaction with isoniazid and the site of formation of Trp • radical appeared to be different depending
on the differences in substrate binding (Colin et al ., 2010).
Wild-type KatG enzyme from Synechocystis sp. strain PCC 6803 and its variants of proximal
triad and distal side Asp152 and Asn153 residues have been studied in their ferric and ferrous state
(at various pH values) by employing resonance Raman spectroscopy and electron absorption. A
comparison of these with CcP revealed that a change in the proximal residues caused a weakening
of haem binding to the protein and so these are important in conferring stability of the protein
architecture. On the other hand, Asp152Ser variation caused an architectural change and the KatG
very much appeared as a CcP. A change in Asn153 to Asp153 resulted in a disruption of the H-bond
with distal His123. It has been further suggested that Asp152, important in H 2 O 2 oxidation reaction,
is expected to be H-bonded to nitrogen atom of Ile248. Any change in the Asp152 and Ile248 H-bond
alters both the stability of haem architecture as well as the interaction between proximal His-Asp
H-bond (Santoni et al ., 2004). Similar conclusions have been drawn by Heering et al . (2002) based
on their studies on distal side variants already described earlier.
Jakopitsch et al . (2005) put forward a proposal for the catalytic mechanism of KatGs and reported
the interconversion of ferrous enzymes, compound II and compound III by the use of wild-type
and Tyr249Phe variants of KatG enzyme of Synechocystis sp. strain PCC 6803. The binding of O 2
to ferrous KatG and Tyr249Phe is reversible and the rate constants are comparable to those of