Abstract
Phenylalanine hydroxylase is a mononuclear non-heme iron protein that uses tetrahydropterin as the source of the two electrons needed to activate dioxygen for the hydroxylation of phenylalanine to tyrosine. Rapid-quench methods have been used to analyze the mechanism of a bacterial phenylalanine hydroxylase from Chromobacterium violaceum. Mössbauer spectra of samples prepared by freeze-quenching the reaction of the enzyme-57Fe(II)-phenylalanine-6- methyltetrahydropterin complex with O2 reveal the accumulation of an intermediate at short reaction times (20-100 ms). The Mössbauer parameters of the intermediate (δ = 0.28 mm/s, and |ΔEQ| = 1.26 mm/s) suggest that it is a high-spin Fe(IV) complex similar to those that have previously been detected in the reactions of other mononuclear Fe(II) hydroxylases, including a tetrahydropterin-dependent tyrosine hydroxylase. Analysis of the tyrosine content of acid-quenched samples from similar reactions establishes that the Fe(IV) intermediate is kinetically competent to be the hydroxylating intermediate. Similar chemical-quench analysis of a reaction allowed to proceed for several turnovers shows a burst of tyrosine formation, consistent with rate-limiting product release. All three data sets can be modeled with a mechanism in which the enzyme-substrate complex reacts with oxygen to form an Fe(IV)=O intermediate with a rate constant of 19 mM -1 s-1, the Fe(IV)=O intermediate hydroxylates phenylalanine with a rate constant of 42 s-1, and rate-limiting product release occurs with a rate constant of 6 s-1 at 5 °C.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1928-1933 |
| Number of pages | 6 |
| Journal | Biochemistry |
| Volume | 50 |
| Issue number | 11 |
| DOIs | |
| State | Published - Mar 22 2011 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
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Evidence for a high-Spin Fe(IV) species in the catalytic cycle of a bacterial phenylalanine hydroxylase. / Panay, Aram Joel; Lee, Michael; Krebs, Carsten; Bollinger, Jr., Joseph M.; Fitzpatrick, Paul F.
In: Biochemistry, Vol. 50, No. 11, 22.03.2011, p. 1928-1933.Research output: Contribution to journal › Article
TY - JOUR
T1 - Evidence for a high-Spin Fe(IV) species in the catalytic cycle of a bacterial phenylalanine hydroxylase
AU - Panay, Aram Joel
AU - Lee, Michael
AU - Krebs, Carsten
AU - Bollinger, Jr., Joseph M.
AU - Fitzpatrick, Paul F.
PY - 2011/3/22
Y1 - 2011/3/22
N2 - Phenylalanine hydroxylase is a mononuclear non-heme iron protein that uses tetrahydropterin as the source of the two electrons needed to activate dioxygen for the hydroxylation of phenylalanine to tyrosine. Rapid-quench methods have been used to analyze the mechanism of a bacterial phenylalanine hydroxylase from Chromobacterium violaceum. Mössbauer spectra of samples prepared by freeze-quenching the reaction of the enzyme-57Fe(II)-phenylalanine-6- methyltetrahydropterin complex with O2 reveal the accumulation of an intermediate at short reaction times (20-100 ms). The Mössbauer parameters of the intermediate (δ = 0.28 mm/s, and |ΔEQ| = 1.26 mm/s) suggest that it is a high-spin Fe(IV) complex similar to those that have previously been detected in the reactions of other mononuclear Fe(II) hydroxylases, including a tetrahydropterin-dependent tyrosine hydroxylase. Analysis of the tyrosine content of acid-quenched samples from similar reactions establishes that the Fe(IV) intermediate is kinetically competent to be the hydroxylating intermediate. Similar chemical-quench analysis of a reaction allowed to proceed for several turnovers shows a burst of tyrosine formation, consistent with rate-limiting product release. All three data sets can be modeled with a mechanism in which the enzyme-substrate complex reacts with oxygen to form an Fe(IV)=O intermediate with a rate constant of 19 mM -1 s-1, the Fe(IV)=O intermediate hydroxylates phenylalanine with a rate constant of 42 s-1, and rate-limiting product release occurs with a rate constant of 6 s-1 at 5 °C.
AB - Phenylalanine hydroxylase is a mononuclear non-heme iron protein that uses tetrahydropterin as the source of the two electrons needed to activate dioxygen for the hydroxylation of phenylalanine to tyrosine. Rapid-quench methods have been used to analyze the mechanism of a bacterial phenylalanine hydroxylase from Chromobacterium violaceum. Mössbauer spectra of samples prepared by freeze-quenching the reaction of the enzyme-57Fe(II)-phenylalanine-6- methyltetrahydropterin complex with O2 reveal the accumulation of an intermediate at short reaction times (20-100 ms). The Mössbauer parameters of the intermediate (δ = 0.28 mm/s, and |ΔEQ| = 1.26 mm/s) suggest that it is a high-spin Fe(IV) complex similar to those that have previously been detected in the reactions of other mononuclear Fe(II) hydroxylases, including a tetrahydropterin-dependent tyrosine hydroxylase. Analysis of the tyrosine content of acid-quenched samples from similar reactions establishes that the Fe(IV) intermediate is kinetically competent to be the hydroxylating intermediate. Similar chemical-quench analysis of a reaction allowed to proceed for several turnovers shows a burst of tyrosine formation, consistent with rate-limiting product release. All three data sets can be modeled with a mechanism in which the enzyme-substrate complex reacts with oxygen to form an Fe(IV)=O intermediate with a rate constant of 19 mM -1 s-1, the Fe(IV)=O intermediate hydroxylates phenylalanine with a rate constant of 42 s-1, and rate-limiting product release occurs with a rate constant of 6 s-1 at 5 °C.
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U2 - 10.1021/bi1019868
DO - 10.1021/bi1019868
M3 - Article
C2 - 21261288
AN - SCOPUS:79952783028
VL - 50
SP - 1928
EP - 1933
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 11
ER -