| Summary: | Haem is an essential molecule that is required for a wide variety of functions in all forms of life. This molecule is required for oxygen transport in humans as a prosthetic group of haemoglobin and myoglobin and is also an integral component of various cytochromes as well as being a cofactor for peroxidases and catalases. Until very recently it was generally accepted that the haem biosynthetic pathway was a conserved process in most organisms that synthesise haem. This dogma has changed with the discovery of two new pathways by which haem can be synthesised. The most recently discovered of these pathways, known as the coproporphyrin-dependent pathway, is utilised by many actinobacteria and firmicutes (including Staphylococcus aureus) and is now thought to be an ancestral pathway to the now more widely distributed classical pathway. In light of this discovery, the current work seeks to examine the biochemical and kinetic properties of the enzymes involved in the terminal stages of haem synthesis in S. aureus (HemY, HemH and HemQ). HemY from S. aureus, which is classically known as a protoporphyrinogen IX oxidase, catalyses the antepenultimate step in a coproporphyrinogen-dependant pathway in vivo was shown for the first time to catalyse the oxidation of coproporphyrinogen III as well as protoporphyrinogen IX, demonstrating that this enzyme can catalyse steps in both classical and coproporphyrin-dependant haem biosynthesis in vitro. Kinetic analyses revealed that the previously-observed stimulation of HemY activity by the terminal pathway enzyme HemQ (a coprohaem decarboxylase) occurred via a peroxidase-mediated mechanism resulting in the generation of superoxide, although this stimulation was not observable when HemY is utilising the native coproporphyrinogen III substrate. This generation of toxic free radicals could explain why HemQ enzymes have not been identified in organisms that synthesise haem via the classical protoporphyrin IX pathway. The HemH enzyme, also known as ferrochelatase, has also been further characterised with its in vivo substrates (Fe2+ and coproporphyrin III), where activity was shown to be diminished by a regulatory metal binding site. The activity of this enzyme with a variety of alternative metal substrates was also assessed, with the enzyme being able to insert metals that are commonly inserted by other ferrochelatases. Kinetic constants for HemQ, a coprohaem decarboxylase, were determined for the first time and HemQ was further assessed for an ability to bind other tetrapyrroles: non-metallated porphyrins were found to bind much tighter than the metalloporphyrins that are the in vivo substrate and product for this enzyme. Together, these intriguing observations have implications for the divergent evolution of haem biosynthesis in ancestral microorganisms and provides new insights into the flux of intermediates through the terminal stages of haem biosynthesis in S. aureus.
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