The Rieske type [2Fe-2 S] cluster of a cytosolic oxygenase from Aspergillus fumigatus

Abstract

Abstract A cytosolic oxygenase enzyme from the mold creating Aspergillus fumigatus has been expressed in Escherichia coli and purified. Mössbauer spectroscopy at 77 K of a 57 Fe-enriched protein (abbreviated as Afox) yields two components with equal relative areas. Component 1 has an isomer shift of δ 1 = 0.26 mms − 1 and a quadrupole splitting of Δ E Q1 = 0.46 mms − 1 . Component 2 shows δ 2 = 0.32 mms − 1 and Δ E Q2 = 0.84 mms − 1 . These parameters are characteristic for a diamagnetic Rieske-type [2Fe-2S] 2+ cluster, in which one ferric ion is coordinated by two cysteine and the two bridging S 2− ions, and the other ferric ion by two histidine residues and the two bridging S 2− ions. High field Mössbauer spectroscopy at 6 K demonstrated that the [2Fe-2S] 2+ cluster has a diamagnetic ground state originating from antiparallel spin coupling of the two high spin ferric ions ( S 1,2 =5/2). Sequence alignment of the amino acids shows that the protein belongs to the class Va Rieske oxygenases, which metabolize quaternary amine substrates such as carnitine, choline or benzalkonium. Using the artificial intelligence tool AlphaFold Colab we have created a structural model showing a binding pocket for the Rieske cluster composed of Cys89, Cys109, His91 and His112, and a binding pocket for a mononuclear iron center. The latter centre was lost during purification and therefore not detected in our Mössbauer spectra. Comparison with carnitine monooxygenase from Acinetobacter baumannii (PDB 6Y8J) suggests a trimeric structure for the A. fumigatus oxygenase. In the trimer the [2Fe-2S] cluster of one monomer is spatially close to the predicted mononuclear iron active site of a neighbouring monomer. Glu212 is predicted to bridge the aforementioned redox centres, which together with the two histidine ligands of the Rieske center play an important role in proton-coupled electron transfer (PCET) in oxygenases.