Biosynthesis and trafficking of heme o and heme a: new structural insights and their implications for reaction mechanisms and prenylated heme transfer

Abstract

Aerobic respiration is a key energy-producing pathway in many prokaryotes and virtually all eukaryotes. The final step of aerobic respiration is most commonly catalyzed by heme-copper oxidases embedded in the cytoplasmic or mitochondrial membrane. The majority of these terminal oxidases contain a prenylated heme (typically heme a or occasionally heme o) in the active site. In addition, many heme-copper oxidases, including mitochondrial cytochrome c oxidases, possess a second heme a cofactor. Despite the critical role of heme a in the electron transport chain, the details of the mechanism by which heme b, the prototypical cellular heme, is converted to heme o and then to heme a remain poorly understood. Recent structural investigations, however, have helped clarify some elements of heme a biosynthesis. In this review, we discuss the insight gained from these advances. In particular, we present a new structural model of heme o synthase (HOS) based on distance restraints from inferred coevolutionary relationships and refined by molecular dynamics simulations that are in good agreement with the experimentally determined structures of HOS homologs. We also analyze the two structures of heme a synthase (HAS) that have recently been solved by other groups. For both HOS and HAS, we discuss the proposed catalytic mechanisms and highlight how new insights into the heme-binding site locations shed light on previously obtained biochemical data. Finally, we explore the implications of the new structural data in the broader context of heme trafficking in the heme a biosynthetic pathway and heme-copper oxidase assembly.

Keywords:

• Cytochrome c oxidase
• cytochrome c oxidase assembly
• heme a synthase
• heme o synthase
• heme-copper oxidases
• heme oxidation
• heme trafficking
• intramembrane aromatic prenyltransferase

Acknowledgments

The authors gratefully acknowledge Prof. Robert Hausinger for his critical reading of this manuscript.

The HOS model is available at https://github.com/feiglab/heme-o-synthase

Disclosure statement

The authors declare that they have no conflicts of interest.

Notes

1 Type-2 HAS can also be referred to as “class D” (Lewin and Hederstedt 2016).

2 HAS from different organisms also typically co-purifies with heme b and heme o or heme a when expressed in E. coli, although the heme type and heme/protein stoichiometry varies (Sakamoto et al. 1999; Lewin and Hederstedt 2006; Mogi 2009b; Hannappel et al. 2011; Zeng et al. 2020).

3 Appending a C-terminal epitope tag to Cox5a causes slight supercomplex destabilization, presumably because the tag disrupts interactions between Cox5a and cytochrome c₁ or Qcr6 in the IMS. See Figure 5D in Herwaldt et al. (2018). The absence of cardiolipin (the bridging lipid that mediates Cox5a-complex III interactions) slightly destabilizes the supercomplexes, while mutagenesis of Cor1 indicates that Cor1-Cox5a interactions are necessary and sufficient for supercomplex formation (Berndtsson et al., 2020).

Additional information

Funding

This work was supported by the NIH NIGMS under grants R35 GM126948 and R01 GM101386.