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The first step in the oxidation of phenols catalyzed by HRP is the formation of a

precursor complex of hydrogen peroxide (or an organic hydroperoxide) with the


enzyme (scheme 2, 4). Next, the the elimination of water leads to the so-called
compound I (scheme 2, 4), with an oxyferryl group in the active center of HRP is
oxidized to Fe(IV) while the porphyrin structure loses one electron. Compound I can
be seen as well as Fe(V) in the active center surrounded be a neutral porphyrin.
Compound 1 oxidizes the firs equivalent of phenol by oxidoreduction. The phenol
coordinates in the active center and is oxideized via one-electron transfer to the
corresponding phenoxy radical cation, while the porphyrin radical cation is reduced
to the neutral porphyrin. After deprotonation of the phenoxy radical cation, the
resulting phenoxy radical leaves the active center. Hence, this mechanism is called
biochemistry an irreversible ping-pong mechanism. The proton of the phenoxy
radical is abstracted by a base, probably distal histidine (His-42) to form the socalled compound 2 (scheme 2, 6). This species oxidizes the second equivalent
phenol. The phenol is oxidized to the phenoxy radical by one-electron transfer to the
fe (IV), so that the native enzyme is regenerated (scheme 2,3). The proton of the
phenol forms water as a leaving group with the oxygen of the oxyferryl group anf
the proton which was bound to the His-42. Thus, in one catalytic cycle, two
equivalents of a phenol are oxidized of hydrogen peroxide producing two phenoxy
radicals and one water molecule (scheme 2).

Mechanism of compound 1 formation


In an initial step, the enzyme turns the hydrogen peroxide into a better nucleophile
by abstraction of one proton to the His-42 and the peroxide coordinates side-on to
the fivefold-coordinated Fe(III) atom (scheme 3,8). Next, a heterolytic cleavage of
the O-O bond is introduced by a push-pull mechanism of two histidine residues in
the active center, the distal His-42, and the proximal His-170. First, the protonated
His-170 facilitates the formation of the iron peroxide bond through the effect of its
positive charge (scheme 3, 9).

Langkah pertama dalam oksidasi fenol dikatalisasi oleh HRP adalah pembentukan
kompleks prekursor hidrogen peroksida (atau hidroperoksida organik) dengan enzim
(skema 2, 4). Selanjutnya, penghapusan air mengarah ke apa yang disebut senyawa
I (skema 2, 4), dengan kelompok oxyferryl di pusat aktif HRP dioksidasi menjadi Fe
(IV) sedangkan struktur porfirin kehilangan satu elektron. Senyawa Saya dapat
dilihat serta Fe (V) di pusat aktif dikelilingi menjadi porfirin netral. Senyawa 1
mengoksidasi setara cemara fenol dengan oxidoreduction. Fenol koordinat di pusat
aktif dan oxideized melalui transfer satu-elektron untuk kation radikal fenoksi yang
sesuai, sedangkan kation radikal porfirin berkurang dengan porfirin netral. Setelah
deprotonasi kation radikal fenoksi, daun radikal pusat aktif fenoksi yang dihasilkan.

Oleh karena itu, mekanisme ini disebut biokimia mekanisme pingpong ireversibel.
Proton dari fenoksi radikal disarikan oleh basa, mungkin distal histidin (Nya-42)
untuk membentuk apa yang disebut senyawa 2 (skema 2, 6). Spesies ini
mengoksidasi fenol setara kedua. Fenol teroksidasi dengan fenoksi radikal dengan
transfer satu elektron ke (IV) fe, sehingga enzim asli dibuat ulang (skema 2,3).
Proton fenol membentuk air sebagai kelompok meninggalkan dengan oksigen dari
kelompok oxyferryl anf proton yang terikat pada-Nya-42. Dengan demikian, dalam
satu siklus katalitik, dua setara fenol teroksidasi hidrogen peroksida memproduksi
dua radikal fenoksi dan satu molekul air (skema 2).

Mekanisme senyawa 1 formasi


Dalam sebuah langkah awal, enzim mengubah hidrogen peroksida menjadi nukleofil
yang lebih baik dengan abstraksi dari satu proton ke-Nya 42 dan peroksida
koordinat sisi-on ke lima kali lipat-dikoordinasikan Fe (III) atom (skema 3,8).
Berikutnya, belahan dada heterolytic dari ikatan OO diperkenalkan oleh mekanisme
push-pull dari dua residu histidin di tengah aktif, distal Nya-42, dan proksimal Nya170. Pertama, terprotonasi Nya-170 memfasilitasi pembentukan peroksida obligasi
besi melalui efek muatan positif (skema 3, 9).

Senyawa 1 kemudian mampu mengoksidasi berbagai mengurangi substrat molekul


(AH) dengan mekanisme yang melibatkan transfer elektron tunggal, dimana pkation radikal pertama habis, yang mengarah ke pembentukan enzim kedua
menengah disebut senyawa II . Senyawa II, yang juga mengandung gugus oxoferryl
(FeIVO), kemudian dikurangi dengan molekul substrat kedua (AH) untuk enzim
besi asli (FeIII). Selama pengurangan satu-elektron ini, kembali besi ferryl ke negara
besi nya, sedangkan oksigen menerima dua proton untuk membentuk molekul air
dan dilepaskan dari heme.
Everse telah mengusulkan bahwa ikatan antara besi dan oksigen di kedua Senyawa
1 dan II bukanlah ikatan ganda konvensional. Bahkan, kecuali untuk panjangnya,
semua pengamatan eksperimental menunjukkan bahwa ikatan oxoferryl mungkin
mirip dengan obligasi heme-O2 di oxy-hemoglobin dan oxy-mioglobin dan juga
untuk ikatan antara atom oksigen dalam ozon, yaitu, bi a radikal, tiga pusat empat
elektron p-ikatan [43].

the reaction starts when H2O2 enters the heme crevice and binds to the heme iron. This
initial interaction between the peroxide and the iron consists in the formation of a ligand
bond between FeIII and one of the peroxide oxygens (the _-oxygen-O_), and in the
subsequent abstraction of a proton by the distal His42. The intermediate enzyme
complex formed is referred in the literature as Compound 0. This compound is very

unstable and has only been detected in cryosolvents at negative temperatures using
peroxidase mutants with low activity.
The next step consists in the heterolytic cleavage of the OO bond. His42 transfers the
abstracted proton to the _-oxygen (O_) and the positively charged guanidinium side
chain of Arg38 stabilises the negative charge that is generated at the O_, promoting the
cleavage of the OO bond. A water molecule is then produced, while the O_ remains
bonded to the heme iron. A fast intramolecular electron transfer then occurs in the
heme active site and the oxygen atom acquires two electrons, one withdrawn from the
iron and the other from the porphyrin ring.
In the mechanism described above, the distal histidine plays the main role as both
proton acceptor from O_ and proton donor to O_. The distal arginine facilitates the
cleavage of the OO bond. Some authors have proposed the involvement of different
residues. If histidine residues are modified with a specific reagent (diethyl
pyrocarbonate), the enzyme is still able to reduce H2O2 and produce Compound I, while
Compound II formation is blocked [83]. It was concluded that His42 was not directly
involved in the formation of Compound I and that the carboxylate side chain of Asp43
could participate in the reaction
The formation of compound I occurs by heterolytic cleavage of the O-O bond of H2O2
through an electron push pull mechanism. The positive distal histidine imidazole (His42) provides the pull and a partially or fully deprotoned proximal histidine (his-170)
provides the push.
The alfa-proton of H2O2 is abstracted by His42, and the positively charged side chain of
arg-38 assists in stabilizing the transition state. Arg is not absolutely essential for
compound 1 formation, but promotes proton transfer to side chain of His42, and
stabilizes the transition state for the heterolytic cleavage of the peroxide O-O bond.