Functional Genomics Investigation of Siroheme and Heme d1 Biosynthesis
Type of DegreePhD Dissertation
Chemistry and Biochemistry
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Many bacteria and archaea grow under anaerobic or microaerophilic conditions. In order for these organisms to survive under these conditions, many utilize nitrate or nitrite as terminal electron acceptors for anaerobic respiration. In addition, some organisms can reduce nitrate or nitrite to ammonia in order to assimilate nitrogen for the synthesis of N-containing biomolecules. The first committed step in both denitrification and/or assimilation is carried out by a nitrite reductase (NIR). There are different types of NIRs and they can be classified by their products (NO vs. NH4+) or by their cofactors, two of which are heme d1 and siroheme. The unique heme d1 cofactor is only found in denitrifying bacteria containing a cytochrome cd1 NIR, and is the site of nitrite reduction. Interestingly, siroheme, the cofactor of some ammonia-forming NIRs, has recently been identified as an intermediate in the biosynthesis of heme d1. In most organisms that produce siroheme and heme d1, a multifunctional enzyme CysG is responsible for the conversion of uroporphyrinogen III to siroheme. However, some nitrate reducing organisms, such as Paracoccus denitrificans and Methanosarcina acetivorans, lack CysG and it is not clear how siroheme is made. In Chapter 2 of this dissertation, we investigate the activity of SirC, a precorrin 2 dehydrogenase from M. acetivorans that is found in the alternative heme biosynthesis (ahb) gene cluster. SirC is homologous to the dehydrogenase/ferrochelatase domain of CysG and it was found that this enzyme converts precorrin 2 to sirohydrochlorin. However, instead of catalyzing iron insertion into sirohydrochlorin, it was found that increasing concentrations of SirC inhibits ferrochelation. We show that multiple tetrapyrroles (uroporphyrinogen III, precorrin 2, and sirohydrochlorin) are able to spontaneously chelate different metal ions, even at physiological concentrations. We therefore propose that SirC protects its product, sirohydrochlorin, from the spontaneous chelation of incorrect metals and passes it on to the next enzyme in the pathway via substrate channeling. Iron is required for the synthesis of siroheme and heme d1. Some organisms (pathogenic bacteria in particular) can acquire the necessary iron from heme using a specific transport system and a cytosolic heme oxygenase (HemO). The denitrifying bacterium P. denitrificans contains a gene cluster with homology to the heme uptake system; however, it lacks the hemO gene. Within this cluster is a gene, pden_1323, that is annotated as a pyridoxamine 5’-phosphate (PMP) oxidase and contains a conserved domain of unknown function (DUF2470). In Chapter 3 we show that this gene is not a PMP oxidase, but rather a non-canonical heme oxygenase. When determining the function of Pden_1323, we also explored the substrate scope of the enzyme and found that deuteroheme and Mn-protoporphyrin IX can be used as substrates. In contrast, protoporphyrin IX was not a viable substrate for the reaction, which supports the proposed mechanism requiring a metal center for catalysis. The iron for the biosynthesis of siroheme and heme d1 in P. denitrificans is proposed to be inserted into sirohydrochlorin by a homolog of CbiX, the cobaltochelatase from the cobalamin biosynthetic pathway. Clustered with the cbiX gene is pden_2333, which encodes a protein with a conserved domain of unknown function (DUF4202). In some species, these two gene are fused together which suggests a role for Pden_2333 in the biosynthesis of siroheme and heme d1. In Chapter 4, we present size exclusion chromatography data that shows that Pden_2333 binds heme and may require it for structural stability and the formation of higher order oligomeric structures. We also were able to show that Pden_2333 is able to load iron into the core of the protein and mobilize it via reduction of the iron. Therefore, we propose that Pden_2333 may be a novel bacterioferritin that provides iron to CbiX for the biosynthesis of siroheme.