Biology Reference
In-Depth Information
assumes signifi cance in the survival of cells where H
2
O
2
is generated by other algae or bacteria in
the ecosystem. The residual peroxidase activity was shown to be light-dependent and due to the
presence of a thioredoxin-specifi c peroxidase (Tichy and Vermaas, 1999).
In the fi rst catalytic reaction cycle of catalases and peroxidases, the ferric enzyme is oxidized
by H
2
O
2
to the redox intermediate compound I with the release of a water molecule (Reaction 1).
This has an oxoferryl center (Fe
IV
=O) in combination with either a porphyrin π-cation radical or an
amino acid radical (R
•+
). Catalse and peroxidase cycles differ in the use of second peroxide molecule.
In catalase reaction, second peroxide molecule is used as a reducing agent for compound I thus
regenerating the native enzyme with the release of O
2
(Reaction 2). This reaction is generally very
faster than compound I formation. In the peroxidase reaction, compound I is transformed in the fi rst
one-electron reduction to compound II containing either oxoferryl (Fe
IV
=O) center or an amino acid
radical (R
•+
) in combination with FeIII (Reaction 3). Compound II is fi nally reduced back to the ferric
peroxidase in a second one-electron reduction (Reaction 4). APxs and CcP cannot perform Reaction
2 and thus can reduce compound I via compound II exclusively thereby oxidizing their substrates
ascorbate and cytochrome
c,
respectively. By contrast catalase-peroxidases perform both peroxidase
(Reactions 1, 3 and 4) and catalytic cycle are active (Jakopitsch
et al
., 2003c).
Peroxidases reduce peroxides by means of one-electron or one two-electron donors. One electron
donors can be aromatic (phenols) or aliphatic (glutathione, NO
-2
anions or metal cations Mn
2+
)
substances and the corresponding oxidation product eventually dimerizes. Two electron donors can
be halides (chloride, bromide, iodide or thiocyanate) with the corresponding oxidation product being
hypophalous acid or hypothiocyanate. That is why the peroxidases that accept electrons from halides
are known as haloperoxidases (irrespective of the protein family that brings about this reaction). The
active site of KatGs consists of a proximal triad (of Asp, Trp and His) and a distal triad (of Arg, Trp
and His) of amino acids and this is similar to the active sites of APx and CcP. Despite this, the Class
I peroxidases differ from one another in many respects. The KatGs have a predominant catalase
activity whereas no such activity has been reported for either APx or CcP. Their reactivities toward
H
2
O
2
, one electron donors and the spectral features of redox intermediates are found to be different.
The KatGs are organized into a two domain monomeric structure with an N-terminal haem domain
that is catalytic and a C-terminal domain that exhibits a high structural and sequence similarity with
N-terminal domain but which is non-functional. But without the C-terminal domain, the N-terminal
domain does not exhibit either catalase or peroxidase activity. The cloned
katG
of
Synechocystis
sp.
strain PCC 6803 was subjected to PCR-based oligonucleotide site-directed mutagenesis to identify the
role of distal amino acid triad (Arg-Trp-His). Overexpression of the respective six genes individually
in
E
.
coli
[BL 21(DE3)pLyS] led to the production of six recombinant proteins where the distal triad
amino acid residues were changed : Arg119Ala, Arg119Asn, Trp122Phe, Trp122Ala, His123Gln and
His123Glu, i.e. Arg119 was exchanged with Ala or Asn; Trp122 was exchanged with Phe or Ala
and His was exchanged with Gln or Glu. The Trp122 mutants completely lost their catalase activity
but the turnover number of catalase activity of 0.02%, 0.03%, 0.5% and 14.6% has been found for
His123Gln, His123Glu, Arg119Asn and Arg119Ala mutant proteins, respectively. The important
role of Trp122 in catalase activity was identifi ed by following the transition of the ferric enzyme to
compound I formation by H
2
O
2
spectroscopically. The distal His-Arg pair is found to be important
in heterolytic cleavage of H
2
O
2
where as the distal Trp is important in the compound I reduction by
H
2
O
2
(Regelsberger
et al
., 2000). Thus the role of conserved Trp residues at the distal haem cavity
site (Trp122) in the formation of compound I was followed by steady-state and stopped-fl ow
spectroscopy. Mutants of
Synechocystis
sp. strain PCC 6803 Trp122Ala and Trp122Phe that completely
lost their catalase activity were not affected in either the formation of compound I or its reduction
