The final step in heme biosynthesis, insertion of ferrous iron into protoporphyrin IX, is catalyzed by protoporphyrin IX ferrochelatase (E.C. 4.99.1.1). It is demonstrated that the pre-steady state human ferrochelatase (R115L) shows 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 ca. 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, converting 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 around 10 times faster than the rate determining step in the steady-state (k cat 0.1 s −1 ). The effect of an additional E343D mutation is apparent at multiple stages in the reaction cycle with a seven fold drop in k cat and three fold drop in k chel . 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. Keywords porphyrin biosynthesis; ferrochelatase; transient kineticsThe final step in heme biosynthesis, insertion of ferrous iron into protoporphyrin IX, is catalyzed by protoporphyrin IX ferrochelatase (protoheme ferro-lyase, E.C. 4.99.1.1, the human enzyme is hereafter referred to as ferrochelatase). The mechanisms of biological iron chelation have proved to be of great interest with a range of studies examining reaction kinetics (1-3), spectroscopy of bound intermediates (4-6), sensitivity of the reaction to structural variation (1,3), and behavior of the enzyme analogues, including antibodies (4,7-9) and both DNA and RNA (10-12) that also catalyze metal ion insertion into porphyrins. Additionally crystal structures of free enzyme (13,14), as well as with bound metal substrate , bound porphyrin substrate (15) and a tight-binding competitive inhibitor (16) should support confident interpretation of the link between structure and function in this system. In fact, controversy remains over the role of individual residues in the reaction mechanism; compare for example the suggested metal ion binding site of Sellers et al. (1) with that proposed by Gora et al. † Funded by the BBSRC (UK).