Evidence for a high-spin Fe(IV) species in the catalytic cycle of a bacterial phenylalanine hydroxylase.
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-(57)Fe(II)-phenylalanine-6-methyltetrahydropterin complex with O(2) 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 |ΔE(Q)| = 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.
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, United States.
Aram Joel
AJ
Panay
Michael
M
Lee
Carsten
C
Krebs
J Martin
JM
Bollinger
Paul F
PF
Fitzpatrick
D001665:Binding Sites; D002384:Catalysis; D002861:Chromobacterium / Q000201:enzymology* / Q000378:metabolism; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D010100:Oxygen / Q000378:metabolism; D010651:Phenylalanine Hydroxylase / Q000737:chemistry* / Q000378:metabolism; D011622:Pterins / Q000737:chemistry / Q000378:metabolism; D015204:Spectroscopy, Mossbauer; D013379:Substrate Specificity
D011622:Pterins; C066317:ferryl iron; C017523:6-methyltetrahydropterin; D007501:Iron; D010651:Phenylalanine Hydroxylase; D010100:Oxygen
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