Evidence for a high-Spin Fe(IV) species in the catalytic cycle of a bacterial phenylalanine hydroxylase

Aram Joel Panay, Michael Lee, Carsten Krebs, Joseph M. Bollinger, Jr., Paul F. Fitzpatrick

Research output: Contribution to journalArticle

57 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)1928-1933
Number of pages6
JournalBiochemistry
Volume50
Issue number11
DOIs
StatePublished - Mar 22 2011

Fingerprint

Phenylalanine Hydroxylase
Phenylalanine
Tyrosine
Rate constants
Nonheme Iron Proteins
Chromobacterium
Oxygen
Hydroxylation
Tyrosine 3-Monooxygenase
Enzymes
Mixed Function Oxygenases
Quenching
Electrons
Acids
Substrates
Chemical analysis
tetrahydropterin

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{4dd17f4bc2e8421684f9c45ec3d20036,
title = "Evidence for a high-Spin Fe(IV) species in the catalytic cycle of a bacterial phenylalanine hydroxylase",
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. M{\"o}ssbauer 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 M{\"o}ssbauer 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.",
author = "Panay, {Aram Joel} and Michael Lee and Carsten Krebs and {Bollinger, Jr.}, {Joseph M.} and Fitzpatrick, {Paul F.}",
year = "2011",
month = "3",
day = "22",
doi = "10.1021/bi1019868",
language = "English (US)",
volume = "50",
pages = "1928--1933",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "11",

}

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 journalArticle

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.

UR - http://www.scopus.com/inward/record.url?scp=79952783028&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79952783028&partnerID=8YFLogxK

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 -