Crystal structure of Escherichia coli class II hybrid cluster protein, HCP, reveals a [4Fe-4S] cluster at the N-terminal protrusion.

Hybrid cluster protein (HCP) is a unique Fe-S-O-type metallocluster-containing enzyme present in many anaerobic organisms and is categorized into three distinct classes (I, II, and III). The class II HCP uniquely utilizes hybrid cluster protein reductase (HCR), unlike the other classes of HCPs. To gain structural insights into the electron transfer system between the class II HCP and HCR, we elucidated the X-ray crystal structure of Escherichia coli HCP (Ec HCP), representing the first report of a class II HCP structure. Surprisingly, Ec HCP was found to harbor a [4Fe-4S] cluster rather than a [2Fe-2S] cluster at the N-terminal Cys-rich region, similar to class I HCPs. It was also found that the Cys-rich motif forms a unique protrusion and that the surrounding charge distributions on the surface of class II Ec HCP are distinct from those of class I HCPs. The functional significance of the Cys-rich region was investigated using an Ec HCP variant (chimeric HCP) containing a class I HCP Cys-rich motif from Desulfovibrio desulfuricans. The biochemical analyses showed that the chimeric HCP lacks the hybrid cluster and the electron-accepting function from HCR despite the formation of the chimeric HCP-HCR complex. Furthermore, HCP-HCR molecular docking analysis suggested that the protrusion area serves as an HCR-binding region. Therefore, the protrusion of the unique Cys-rich motif and the surrounding area of class II HCP are likely important for maturation of Ec HCP and orienting HCR onto the surface of HCP to facilitate electron transfer in the HCP-HCR complex.

Structure of sirohydrochlorin ferrochelatase SirB: the last of the structures of the class II chelatase family.

The crystal structure of Bacillus subtilis SirB, which catalyses the insertion of Fe2+ into the substrate sirohydrochlorin (SHC) in siroheme biosynthesis, is reported herein as the last of the structures of class II chelatases. The structure of SirB with Co2+ showed that the active site of SirB is located at the N-terminal domain with metal-binding amino acid residues His10, Glu43, and His76, which was also predicted for CbiX, but is distinct from the C-terminal active sites of CbiK and HemH. The biosynthetic model reactions using SirB, Co2+ and uroporphyrin I or protoporphyrin IX as a SHC analogue revealed that SirB showed chelatase activity for uroporphyrin I, but not for protoporphyrin IX. Simulations of tetrapyrroles docking to SirB provided an insight into its tetrapyrrole substrate recognition: SHC and uroporphyrin I were suitably bound beside the Co2+ ion-binding site at the active site cavity; protoporphyrin IX was also docked to the active site but its orientation was different from those of the other two tetrapyrroles. Summarizing the present data, it was proposed that the key structural features for substrate recognition of SirB could be the hydrophobic area at the active site as well as the substituents of the tetrapyrroles.