STRESS RESPONSES - Cyanobacteria - page 690

to compound II in presence of O-dianisidine or pyrogallol. These observations signify that indole
ring of distal tryptophan is essential for two-electron reduction of compound I by H 2 O 2 and not for
compound I formation (Regelsberger et al ., 2001).
The Synechocystis catalase-peroxidase enzyme also proved to be a good haloperoxidase capable of
oxidizing chloride, bromide and iodide. This was tested by the halogenation of monochlorodimedon
(MCD) by chloride, bromide and iodide by the wild-type and variant Arg119Ala, Trp122Phe and
Trp122Ala enzymes. As mentioned earlier, the Trp122 mutated to Phe and Ala lost the catalase as
well as halogenation activities but with an overall increase in peroxidase activity. This indicates
that the same redox intermediate is involved in H 2 O 2 and halide oxidation (Jakoptisch et al ., 2001).
Recombinant wild-type KatG enzyme and the distal side variants (Arg119Ala, Arg119Asn, Trp122Phe,
Trp122Ala, His123Gln and His123Glu) from Synechocystis sp. strain PCC 6803 have been subjected
to UV-Vis and resonance Raman spectroscopy to fi nd out distal cavity interactions. These involve
H-bond networks that connect the distal side residues with proximal side of the haem pocket.
The distal mutations affected the Fe haem coordination state and also the stability of the haem
architecture (Heering et al ., 2002). Apart from the distal triad of the active site, the importance of
distal Asp152 residue was recognized. The side chain carboxyl group of Asp152 is 7.8 Å away from
the haem iron and is hydrogen bonded to two water molecules and a KatG-specifi c loop. Mutants
of this distal Asp152 were generated by site-directed mutagenesis and Asp152Asn, Asp152Ser, and
Asp152Trp variants showed a reduced catalase activity with the turnover number of 2.7%, 5.7% and
0.6% of the wild-type, respectively. The variants showed enhanced (2.7 times) peroxidase activity
than wild-type and another variant Pro151Ala enzymes. The variant enzymes showed an altered
pH profi le and it was possible to determine spectroscopically the redox intermediate compound
by H 2 O 2 . In the Asp152 variants the reactivity of compound toward aromatic one electron donors
was enhanced with a decrease in the reactivity to H 2 O 2 . A mechanism of H 2 O 2 oxidation different
than that of monofunctional catalases involving the distal Trp122 and Asp152 has been predicted
(Jakopitsch et al ., 2003a). Since the distal histidine (His123) forms a hydrogen bond with the adjacent
asparagine (Asn153), the role of this hydrogen bond was investigated in the bifunctional activity of
KatGs. Replacement of asparagine with either Ala (Asn153Ala) or aspartic acid (Asn153Asp) did
not affect the peroxidase activity. However, the turnover number of catalase activity was found to be
16.5% and 6% of that of wild-type in Asn153Asp and Asn153Ala variants, respectively. This shows
that Asn153 is an important part of the extended H-bond network of Kat G and it seems to provide
optimal conditions for binding and oxidation of the second molecule of H 2 O 2 necessary in the catalase
reaction (Jakopitsch et al ., 2003b). Investigating the role of tyrosine, a distal side residue (Tyr249),
Jakopitsch et al. (2003c) found that the bifunctional KatG completely lost its catalase activity with the
replacement of tyrosine by phenylalanine (Tyr249Phe) but retained its peroxidase activity. Similar
to the wild-type enzyme, Tyr249Phe variant enzyme could use a range of substrates (O-dianisidine,
pyrogallol, guaiacol, tyrosine and ascorbate) for its peroxidase activity. The formation of redox
intermediate compound I has been demonstrated spectroscopically by the addition of equimolar H 2 O 2
by the variant enzyme. Further, for the fi rst time the transition from compund I to an oxoferryl-like
compund II was detected and the rate constants for the formation of both compounds determined
by the sequential stopped-fl ow technique.
The formation of protein-based radicals was demonstrated in wild-type KatG enzyme of
Synechocystis sp. strain PCC 6803 using a multifrequency EPR investigation (Ivancich et al ., 2003).
EPR signal for the formation of compound I intermediate [FeIV=O por ++ ] and Tyr • and Trp • radicals
was detected. The Trp • radical formation was supposed to be from Trp106, which is located at a
distance of 7-8 Å from haem propionate group. Further it has been suggested that the extensive