Ferrochelatase reaction mechanism

Although resonance Raman [44] and fluorescence data [47] demonstrate that porphyrin binding can occur in the absence of metal, kinetic studies of bovine ferrochelatase appear to be consistent with an ordered bi-bi sequential mechanism, in which iron binding occurs prior to that of porphyrin [39, 42], In contrast, Labbe-Bois and Camadro [11] and Rossi et al. [48] proposed a random bi-bi mechanism in which each substrate binds randomly to the enzyme and the binding of the first substrate does not affect the binding of the second one. Which of these two proposed kinetic mechanisms is correct for ferrochelatase remains an open question. 

The early observation that ferric iron could not be used as substrate by ferrochela-tase [3], led to the development of an assay for enzyme activity determination based on the use of DTT (dithiothreitol) to maintain the iron ions in the reduced +2 form. This requirement for DTT in the enzymatic assay was assumed to be an essential feature, and accordingly, either DTT or some other reducing agent was always included in the buffers used in ferrochelatase purification as well as in the activity assay [9, 49]. However, as Porra et al. [50] and Punekar and Gokhale [51] pointed 

3 Ferrochelatase a new iron sulfur center-containing enzyme 

Previously, Tangeras [52] had proposed that a pool of iron in the inner mitochondrial compartment (1 nmol mg of protein) is available to ferrochelatase for heme formation. A soluble component of that compartment could maintain sufficient ferrous iron in equilibrium with ferric iron, allowing the observed rate of 0.3 nmol of heme formation per hour, which corresponds to an amount of about five times that necessary for the turnover of hemoproteins in hepatocytes [53]. 

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Perhaps the best-characterized example of this mechanism involves the synthesis of heme cofactors and their subsequent incorporation into various hemoproteins (see Iron Heme Proteins Electron Transport). Succinctly, enzyme-catalyzed reactions convert either succinyl-CoA or glutamate into 5-ammolevulinic acid. This molecule is further converted through a series of intermediates to form protoporphyrin IX, the metal-ffee cofactor, into which Fe is inserted by ferrochelatase. Analogous reactions are required for the synthesis of other tetrapyrrole macrocycles such as the cobalamins (see Cobalt Bu Enzymes Coenzymes), various types of chlorophylls, and the methanogen coenzyme F430 (containing Co, Mg, or Ni, respectively). Co- and Mg-chelatases have been described for insertion of these metals into the appropriate tetrapyrrolic ring structures. ... 

Figure 3-2 Proposed mechanism for the ferrochelatase catalyzed reaction. Porphyrin distortion induced by metal binding leads to a domed transition state with concomitant porphyrin-proton release (Adapted from [41]). FC, ferrochelatase M, substrate metal ion.

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