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. 2007 Jul 10;46(27):8121-7.
doi: 10.1021/bi602418e. Epub 2007 Jun 13.

Direct measurement of metal ion chelation in the active site of human ferrochelatase

M Hoggins  1 , H A Dailey, C N Hunter, J D Reid
Affiliations

Direct measurement of metal ion chelation in the active site of human ferrochelatase

M Hoggins et al. Biochemistry. .

Abstract

The final step in heme biosynthesis, insertion of ferrous iron into protoporphyrin IX, is catalyzed by protoporphyrin IX ferrochelatase (EC 4.99.1.1). We demonstrate that pre-steady state human ferrochelatase (R115L) exhibits a stoichiometric burst of product formation and substrate consumption, consistent with a rate-determining step following metal ion chelation. Detailed analysis shows that chelation requires at least two steps, rapid binding followed by a slower (k approximately 1 s-1) irreversible step, provisionally assigned to metal ion chelation. Comparison with steady state data reveals that the rate-determining step in the overall reaction, conversion of free porphyrin to free metalloporphyrin, occurs after chelation and is most probably product release. We have measured rate constants for significant steps on the enzyme and demonstrate that metal ion chelation, with a rate constant of 0.96 s-1, is approximately 10 times faster than the rate-determining step in the steady state (kcat = 0.1 s-1). The effect of an additional E343D mutation is apparent at multiple stages in the reaction cycle with a 7-fold decrease in kcat and a 3-fold decrease in kchel. This conservative mutation primarily affects events occurring after metal ion chelation. Further evaluation of structure-function data on site-directed mutants will therefore require both steady state and pre-steady state approaches.

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Figures

Scheme 1
Scheme 1
Two step substrate binding to an enzyme, E.
Scheme 2
Scheme 2
A minimal model for the ferrochelatase reaction.
Figure 1
Figure 1
Initial rates of ferrochelatase catalyzed insertion of iron into deuteroporphyrin at pH 8.1, 100mM TRIS-HCl, 250 μM Fe2+, 1mM β mercaptoethanol, 0.5% (v/v) Tween 20, 30°C. Top, 0.17 μM R115L ferrochelatase, deuteroporphyrin concentrations were A 1.8 μM, B 3.6 μM, C 6.3 μM, D 9.9 μM, E 15.3 μM, F 29.7 μM, and G 49.6 μM. The points are experimental and the theoretical lines are described by equation 1 with characterizing parameters KmFe 7.7 ± 1.4 μM, KmDIX 5.5 ± 0.5 μM, KFe.DIX 30.54 ± 8.01 μM2, V 1.21 μMmin−1. Bottom, 0.16 μM R115L, E343D ferrochelatase, deuteroporphyrin concentrations were A 1.9 μM, B 4.0 μM, C 5.9 μM, D 9.9 μM, E 14.9 μM, F 29.7 μM, and G 49.7 μM. The points are experimental and the theoretical lines are described by equation 1 with characterizing parameters KmFe 8.2 ± 1.0 μM, KmDIX 5.8 ± 0.8 μM, KFe.DIX 34.8 ± 8.3 μM2, V 0.16 ± 0.01 μMmin−1.
Figure 2
Figure 2
Secondary plots showing the iron dependence of the observed kinetic parameters. The points are the apparent kinetic parameters that arise from fitting the Michaelis-Menten equation to individual vi vs. [S] curves and the lines are described by equation 1 (Vapp filled squares, V/Kapp filled circles) with characterizing parameters, (top) for R115L ferrochelatase KmFe 7.7 ± 1. 4 μM, KmDIX 5.5 ± 0.5 μM, KFe.DIX 30.54 ± 8.01 μM2, V 1.21 μMmin−1 and (bottom) R115L, E343D ferrochelatase KmFe 8.2 ± 1.0 μM, KmDIX 5.8 ± 0.8 μM, KFe.DIX 34.8 ± 8.3 μM2, V 0.16 ± 0.01 μMmin−1.
Figure 3
Figure 3
Time-course of DIX (25 μM) consumption by ferrochelatase (0.5 μM) at pH 8.1, 100mM TRIS-HCl, 250 μM Fe2+, 1mM β mercaptoethanol, 0.5% (v/v) Tween 20, 30°C.
Figure 4
Figure 4
The dependence of kobs for the reaction of R115L human ferrochelatase (0.5 μM) on deuteroporphyrin concentration at pH 8.1, 100mM TRIS-HCl, 250 μM Fe2+, 1mM β mercaptoethanol, 0.5% (v/v) Tween 20, 30°C. The points are experimental and the theoretical line is described by equation 3 and characterizing parameters kchel 0.97 ± 0.05 s−1 and KsDIX 16.3 ± 1.9 μM.
Figure 5
Figure 5
The dependence of kobs for the reaction of R115L human ferrochelatase (0.5 μM) on Fe2+ concentration at pH 8.1, 100mM TRIS-HCl, 50 μM DIX, 1mM β metcaptoethanol, 0.5% (v/v) Tween 20, 30°C. The points are experimental and the theoretical line is described by equation 3 and characterizing parameters kchel 0.96 ± 0.05 s−1 and KsFe 79.1 ± 11.2 μM.
Figure 6
Figure 6
The dependence of kobs for the reaction of E343D, R115L human ferrochelatase (0.5 μM) on deuteroporphyrin concentration at pH 8.1, 100mM TRIS-HCl, 350 μM Fe2+, 1mM β mercaptoethanol, 0.5% (v/v) Tween 20, 30°C. The points are experimental and the theoretical line is described by equation 3 and characterizing parameters kchel 0.35 ± 0.3 s−1 and KsDIX 18.6 ± 4.2 μM.
Figure 7
Figure 7
The dependence of kobs for the reaction of E343D, R115L human ferrochelatase (0.5 μM) on Fe2+ concentration at pH 8.1, 100mM TRIS-HCl, 50 μM DIX, 1mM β metcaptoethanol, 0.5% (v/v) Tween 20, 30°C. The points are experimental and the theoretical line is described by equation 3 and characterizing parameters kchel 0.30 ± 0.03 s−1 and KsFe 97.6 ± 15.7 μM.
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