A new class of [2Fe-2S]-cluster-containing protoporphyrin (IX) ferrochelatases

Abstract

Protoporphyrin (IX) ferrochelatase catalyses the insertion of ferrous iron into protoporphyrin IX to form haem. These ferrochelatases exist as monomers and dimers, both with and without [2Fe-2S] clusters. The motifs for [2Fe-2S] cluster co-ordination are varied, but in all cases previously reported, three of the four cysteine ligands are present in the 30 C-terminal residues and the fourth ligand is internal. In the present study, we demonstrate that a group of micro-organisms exist which possess protoporphyrin (IX) ferrochelatases containing [2Fe-2S] clusters that are co-ordinated by a group of four cysteine residues contained in an internal amino acid segment of approx. 20 residues in length. This suggests that these ferrochelatases have evolved along a different lineage than other bacterial protoporphyrin (IX) ferrochelatases. For example, Myxococcus xanthus protoporphyrin (IX) ferrochelatase ligates a [2Fe-2S] cluster via cysteine residues present in an internal segment. Site-directed mutagenesis of this ferrochelatase demonstrates that changing one cysteine ligand into serine results in loss of the cluster, but unlike eukaryotic protoporphyrin (IX) ferrochelatases, this enzyme retains its activity. These data support a role for the [2Fe-2S] cluster in iron affinity, and strongly suggest convergent evolution of this feature in prokaryotes.

Figure 1. Multiple sequence alignment of protoporphyrin (IX) ferrochelatases

The black shading indicates a 90% sequence identity and the grey shading indicates where 90% of the residues are functionally similar. The solid boxes highlight potential conserved cysteine residues in the subset of ferrochelatases containing the ‘cysteine-rich insertion’. *, the cysteine ligands to the iron-sulphur cluster in human ferrochelatase. The broken-line boxes indicate the primary sequence of human ferrochelatase that is shaded in blue in Figure 5.

Figure 2. UV absorption spectra of protoporphyrin (IX) ferrochelatases

Absorption spectra of wild-type (solid line) and C219S mutant (dashed line) ferrochelatases from M. xanthus, and C341S mutant (dotted line) ferrochelatase from C. crescentus. The peak heights have been normalized for clarity.

Figure 3. V versus [Fe²⁺] curves for M. xanthus wild-type (■) and C219S mutant (●) protoporphyrin (IX) ferrochelatases

Both datasets were fitted to single rectangular hyperbolae, but only the datapoints below 25 μM Fe²⁺ were used for the regression analysis of the C219S mutant data. The k_cat and K_app^Fe values for the wild-type enzyme were 4.9±0.3 min⁻¹ and 6.5±1.7 μM respectively. The k_cat and K_app^Fe values for the C219S mutant enzyme were 2.0±0.4 min⁻¹ and 13.9±3.0 μM respectively.

Figure 4. V versus [Fe²⁺] curves for C. crescentus wild-type (■) and C341S mutant (●) protoporphyrin (IX) ferrochelatases

Both datasets were fitted to single rectangular hyperbolae. The k_cat and K_app^Fe values for the wild-type enzyme were 16.0±1.2 min⁻¹ and 11.0±2.3 μM respectively. The k_cat and K_app^Fe values for the C431S mutant enzyme were 3.8±0.1 and 2.8±0.5 μM respectively.

Figure 5. Ribbon diagram of the human protoporphyrin (IX) ferrochelatase structure showing the spatial orientation of the [2Fe-2S] cluster and the cysteine residues co-ordinating it

The blue shading indicates the region of the human enzyme that corresponds to the cysteine rich insertion in some bacterial protoporphyrin (IX) ferrochelatases (broken-line box in Figure 1). The C-terminal extension found in human, but not these bacterial enzymes, is shown in green. The iron-sulphur cluster is shown in red.