The heme biosynthetic enzyme coproporphyrinogen III oxidase and the heme chaperone HemW from bacteria

Abstract

The thesis focused on a novel enzyme of heme biosynthesis (YtpQ) and the first cytoplasmic heme chaperone HemW. The putative coproporphyrinogen III oxidase YtpQ from Bacillus megaterium was recombinantly produced. Subsequent analysis of its enzymatic activity revealed that YtpQ is a true coproporphyrinogen III oxidase converting coproporphyrinogen III to coproporphyrin III. YtpQ is able to catalyzes this reaction in the absence of oxygen using menadione as an alternative electron acceptor potentially linking this step of heme biosynthesis to the respiratory chain. Additionally, it was shown that FAD serves as an cofactor for YtpQ. Kinetic analysis of YtpQ resulted in a KM of 7.9 µmol/L and a kcat of 0.895 min-1. In summary, it was shown that YtpQ from the Gram-positive bacterium B. megaterium is an oxygen-independent coproporphyrinogen III oxidase representing an anaerobe alternative to CgoX during the recently described coproporphyrin-dependent heme b biosynthesis. The heme chaperone HemW from Escherichia coli was functionally characterized. Growth behavior analysis showed a slight phenotype of a hemW deletion mutant strain during anaerobic growth. In order to get further insight into the influence of HemW´s [4Fe-4S] cluster a HemW iron-sulfur cluster deficient variant was generated. A gel permeation chromatography of HemW and the iron-sulfur cluster deficient HemW variant clearly demonstrated that the iron-sulfur cluster is required for dimerization. However, the iron-sulfur cluster was not essential for heme binding as demonstrated via UV-Vis spectroscopy and heme staining. Using the bacterial adenylate cyclase-based two-hybrid system bacterioferritins BfrA and BfrB as well as E. coli nitrate reductase (NarGHI) subunit NarI were identified as HemW interaction partners. A subsequent heme transfer assay using heme deficient NarGHI containing membrane vesicles clearly showed the ability of HemW to transfer bound heme to its target NarI and to restore NarGHI activity. This was not true for the iron-sulfur cluster deficient variant. In conclusion it was demonstrated that HemW serves as a heme chaperone navigating heme from the heme-binding protein bacterioferritin to the heme enzyme nitrate reductase. The presence of the iron sulfur cluster influences HemW dimerization and heme transfer to target proteins but does not influence HemW´s heme binding ability. The potential involvement of a radical SAM mechanism remains to be determined.