Conversion of a heme-based oxygen sensor to a heme oxygenase by hydrogen sulfide: effects of mutations in the heme distal side of a heme-based oxygen sensor phosphodiesterase (Ec DOS)

Abstract

The heme-based oxygen-sensor phosphodiesterase from Escherichia coli (Ec DOS), is composed of an N-terminal heme-bound oxygen sensing domain and a C-terminal catalytic domain. Oxygen (O2) binding to the heme Fe(II) complex in Ec DOS substantially enhances catalysis. Addition of hydrogen sulfide (H2S) to the heme Fe(III) complex in Ec DOS also remarkably stimulates catalysis in part due to the heme Fe(III)-SH and heme Fe(II)-O2 complexes formed by H2S. In this study, we examined the roles of the heme distal amino acids, M95 (the axial ligand of the heme Fe(II) complex) and R97 (the O2 binding site in the heme Fe(II)-O2 complex) of the isolated heme-binding domain of Ec DOS (Ec DOS-PAS) in the binding of H2S under aerobic conditions. Interestingly, R97A and R97I mutant proteins formed an oxygen-incorporated modified heme, verdoheme, following addition of H2S combined with H2O2 generated by the reactions. Time-dependent mass spectroscopic data corroborated the findings. In contrast, H2S did not interact with the heme Fe(III) complex of M95H and R97E mutants. Thus, M95 and/or R97 on the heme distal side in Ec DOS-PAS significantly contribute to the interaction of H2S with the Fe(III) heme complex and also to the modification of the heme Fe(III) complex with reactive oxygen species. Importantly, mutations of the O2 binding site of the heme protein converted its function from oxygen sensor to that of a heme oxygenase. This study establishes the novel role of H2S in modifying the heme iron complex to form verdoheme with the aid of reactive oxygen species.

Hydrogen sulfide activation in hemeproteins: The sulfheme scenario

“…Moreover, as Table 1 shows, sulfheme was not detected in the HbI Gln/Val, Gln/Asn and Gln/Arg mutants, while the product was only observed in the Gln/His variant [14, 18]. Similarly, it was shown recently that the native Ec DOS-PAS heme sensor, which has Met and Arg in its distal site, does not form the sulfheme complex, supporting the role His in sulfheme formation [86]. Mutations of these residues by Ala, Ile, Leu and Glu did not induce formation of sulfheme, instead a verdoheme product was observed in the Arg/Ala and Arg/Ile mutants.…”

“…Verdoheme is a modified heme that has oxygen incorporated in the α-meso heme position. It is known for being one of the heme degradation steps in heme oxygenase [86]. Thus, it is evident that amino acids with: (i) non-polar aliphatic groups like glycine, alanine, valine and isoleucine, (ii) polar uncharged groups like glutamine and asparagine, (iii) aromatic group like phenylalanine and tyrosine, and (iv) positively charged groups like lysine and arginine, are not involved in the synthesis of the sulfheme species.…”

“…This exclusive behavior can be attributed to the distance between the His and the heme iron, which is shorter in LPO (4.94 Å), as opposed to HRP (6.00 Å) (Figure 5a and 5d). On the other hand, the His residue in the Ec DOS-PAS Met95His mutant is coordinated to the heme iron, which blocks H 2 O 2 binding and sulfheme formation [86]. Clearly, this shows that the orientation and position of the His residue at the heme active site are crucial for the synthesis of the sulfheme product.…”

The Heme-Based Oxygen-Sensor Phosphodiesterase Ec DOS (DosP): Structure-Function Relationships

“…Accordingly, the autoxidation rates of mutants at the R97 residue are very fast [ 33 ]. Upon addition of H 2 S to the R97A and R97I mutants of Ec DOS-PAS-A-heme Fe(III), an O 2 -incorporated modified heme is formed with absorption band characteristic of verdoheme [ 40 ]. Time-dependent mass spectroscopic analyses confirmed that this treatment resulted in the formation of verdoheme and a trace of biliverdin, suggesting that H 2 O 2 or superoxide anion generated by these mutants would help to form verdoheme and biliverdin.…”

An oxidative N-demethylase reveals PAS transition from ubiquitous sensor to enzyme

“…Our data confirm that the PAS fold can accommodate enzymatic activity, which suggests other PAS enzymes are likely to exist. Furthermore, it points to the fact that existing PAS domains can be evolved to catalyse reactions: engineered variants of a PAS oxygen sensor have been shown to display heme oxygenase activity [25]. N--dealkylation is a widespread reaction, catalysed by cytochromes P450 [12], FAD/quinone amine oxidases [26,27] and α--ketoglutarate--dependent Fe oxygenases [28].…”