Variable composition of heme oxygenases with different regiospecificities in Pseudomonas species.

Heme oxygenases (HO) degrade heme yielding iron, carbon monoxide and one of four possible biliverdin (BV) isomers. Pseudomonas aeruginosa PAO1 is thus far the only organism to contain two HOs with different regiospecificities: BphO and PigA. While BphO cleaves heme to exclusively yield BV IXα, PigA produces the BV isomers IXß and IXδ. We bioinformatically identified putative HOs in diverse Pseudomonas strains, tested their enzymatic functionality and determined their regiospecificity. Surprisingly, even high amino acid sequence identities to the P. aeruginosa HOs were not sufficient to correctly predict the HO regiospecificity in all cases. Based on our results, Pseudomonas strains differ in their HO composition containing either BphO or PigA or both HO types. Concomitantly with the existence of bphO is the occurrence of at least one gene encoding a bacterial phytochrome implying that only BV IXα is the sufficient phytochrome chromophore. In contrast, pigA genes are organized in gene clusters associated with iron utilization implying a role of PigA in iron acquisition. However, at least in strains containing no PigA this function maybe fulfilled by BphO. Only a combination of homology searches and analyses of genetic environments is appropriate for a reliable prediction of the regiospecificity of Pseudomonas HOs.

Characterization of the haem oxygenase protein family in Arabidopsis thaliana reveals a diversity of functions.

HOs (haem oxygenases) catalyse the oxidative cleavage of haem to BV (biliverdin), iron and carbon monoxide. In plants, the product of the reaction is BV IXalpha, the precursor of the PHY (phytochrome) chromophore and is thus essential for proper photomorphogenesis. Arabidopsis thaliana contains one major biochemically characterized HO (HY1) and three additional putative HOs (HO2, HO3 and HO4). All four proteins are encoded in the nucleus but contain chloroplast translocation sequences at their N-termini. The transit peptides of all four proteins are sufficient for chloroplast translocalization as shown by GFP (green fluorescent protein) reporter gene fusions. Overall, all four proteins can be divided into two subfamilies: HO1 and HO2. Here we show that all members of the HO1 subfamily (HY1, HO3 and HO4) are active monomeric HOs and can convert haem to BV IXalpha using spinach Fd (ferredoxin) as an electron donor. Addition of a second electron donor, such as ascorbate, led to a 10-fold increase in the haem conversion rate. Furthermore, haem turnover is also promoted by light when spinach thylakoids are present. All HO1 family members displayed similar kinetic parameters indicating they all have a possible involvement in PHY chromophore biosynthesis. HO2 did not yield sufficient amounts of soluble protein and therefore required the construction of a synthetic gene adapted to the codon usage of Escherichia coli. HO2 is unable to bind or degrade haem and therefore it is not a haem oxygenase. However, HO2 shows strong binding of proto IX (protoporphyrin IX), a precursor for both haem and chlorophyll biosynthesis. A possible function of HO2 in the regulation of tetrapyrrole metabolism is discussed.