Direct spectroscopic detection of a C-H-cleaving high-spin Fe(IV) complex in a prolyl-4-hydroxylase

Abstract

The Fe(II)- and alpha-ketoglutarate (alphaKG)-dependent dioxygenases use mononuclear nonheme iron centers to effect hydroxylation of their substrates and decarboxylation of their cosubstrate, alphaKG, to CO(2) and succinate. Our recent dissection of the mechanism of taurine:alphaKG dioxygenase (TauD), a member of this enzyme family, revealed that two transient complexes accumulate during catalysis in the presence of saturating substrates. The first complex contains the long-postulated C-H-cleaving Fe(IV)-oxo intermediate, J, and the second is an enzyme.product(s) complex. Here, we demonstrate the accumulation of two transient complexes in the reaction of a prolyl-4-hydroxylase (P4H), a functional homologue of human alphaKG-dependent dioxygenases with essential roles in collagen biosynthesis and oxygen sensing. The kinetic and spectroscopic properties of these two P4H complexes suggest that they are homologues of the TauD intermediates. Most notably, the first exhibits optical absorption and Mössbauer spectra similar to those of J and, like J, a large substrate deuterium kinetic isotope on its decay. The close correspondence of the accumulating states in the P4H and TauD reactions supports the hypothesis of a conserved mechanism for substrate hydroxylation by enzymes in this family.

Scheme 1.

Consensus mechanism for oxygen activation by the Fe(II)- and αKG-dependent dioxygenases. The states labeled J and V have been trapped and characterized for TauD (–35) and P4H (this work).

Fig. 1.

Spectrophotometrically monitored titration of αKG into a solution of the P4H·Fe(II) complex at 5°C in the absence of O₂. The starting protein solution contained 650 μM P4H and 500 μM Fe(II) in P4H buffer (described in Experimental Methods). The spectra shown correspond to αKG concentrations of 0.17 mM (circles), 0.33 mM (squares), 0.54 mM (diamonds), and 0.98 mM (triangles). The points in Inset depict the change of absorbance at 520 nm. The solid line is a fit yielding a K_d of 27 ± 6 μM.

Fig. 2.

SF absorption kinetic traces from reactions of P4H·Fe(II)·αKG [±(Pro-Ala-Pro-Lys)₃] containing either unlabeled (Pro-Ala-Pro-Lys)₃ or deuteriated substrate (d₇-Pro-Ala-d₇-Pro-Lys)₃ with O₂. Final reaction conditions were 0.75 mM P4H, 0.50 mM Fe(II), 5 mM αKG, 5 mM ascorbate, 5 mM substrate, and 0.2 mM O₂. (Upper) Absorbance at 520 nm for the reactions lacking any peptide substrate (squares) and with (Pro-Ala-Pro-Lys)₃ (open circles). The solid line is a fit of the equation given in Experimental Methods to the data. (Lower) Absorbance at 320 nm in the reactions of (Pro-Ala-Pro-Lys)₃ (open circles) and (d₇-Pro-Ala-d₇-Pro-Lys)₃ (closed circles). The solid lines are fits, as described in Experimental Methods.

Fig. 3.

4.2-K/40-mT Mössbauer spectra of selected samples of P4H. (Left) O₂-free P4H·Fe(II)·αKG·(Pro-Ala-Pro-Lys)₃ complex (Top) and 20-ms freeze-quenched samples from the reaction of this complex with O₂ at 5°C [Middle, deuteriated (d₇-Pro-Ala-d₇-Pro-Lys)₃; Bottom, unlabeled (Pro-Ala-Pro-Lys)₃]. The solid line in the middle spectrum is the contribution of the Fe(IV) intermediate (38%) simulated according to the parameters quoted in the text. The solid line in Bottom is the contribution of the P4H·Fe(II)·αKG·(Pro-Ala-Pro-Lys)₃ complex (62%). (Right) Comparison of derived spectra of the three accumulating states from the P4H (vertical lines) and TauD (solid lines) catalytic cycles: quaternary complex (Top), Fe(IV) intermediate (Middle), and product(s) complex (Bottom). For the 20-ms samples, the concentrations after mixing were 1.4 mM P4H, 1.25 mM Fe(II), 5 mM αKG, 5 mM ascorbate, 3.3 mM (Pro-Ala-Pro-Lys)₃ (unlabeled or deuteriated), and 1.2 mM O₂.

Fig. 4.

Kinetics of the P4H reaction by SF absorption and FQ Mössbauer. Left, 4.2-K/40-mT Mössbauer spectra of samples quenched at 20 ms, 68 ms, 160 ms, and 370 ms. The solid line in the top spectrum is the derived spectrum of J. Right, Comparison of the deduced concentration of J with the A₃₂₀ kinetic trace from the SF experiment with identical reaction conditions. Error bars on the concentrations of J deduced from Mössbauer spectra reflect the uncertainty of ±3% of the absorption area of the spectra (ordinate) and the uncertainty of the reaction time of ±5 ms (abscissa). Optimal agreement between concentration and absorbance required Δε₃₂₀ = 1,500 M⁻¹·cm⁻¹ for the Fe(IV) intermediate. Concentrations were 1 mM P4H, 0.75 mM Fe(II), 5 mM αKG, 5 mM ascorbate, 5 mM (d₇-Pro-Ala-d₇-Pro-Lys)₃, and 0.65 mM O₂.

Fig. 5.

4.2-K/variable-field Mössbauer spectra of the 20-ms sample from the (d₇-Pro-Ala-d₇-Pro-Lys)₃ reaction shown in Fig. 3. (Left) Experimental spectrum (vertical lines) and spectrum of O₂-free control, scaled to 62% of the total intensity (solid line). (Right) Derived spectra of the Fe(IV) intermediate from P4H (vertical lines) and spin Hamiltonian simulations (solid lines) according to the parameters given in Table 1.