Insights into the catalytic mechanism of type VI sulfide:quinone oxidoreductases

Abstract

Sulfide oxidation is catalyzed by ancient membrane-bound sulfide:quinone oxidoreductases (SQR) which are classified into six different types. For catalysis of sulfide oxidation, all SQRs require FAD cofactor and a redoxactive centre in the active site, usually formed between conserved essential cysteines. SQRs of different types have variation in the number and position of cysteines, highlighting the potential for diverse catalytic mechanisms. The photosynthetic purple sulfur bacterium, Thiocapsa roseopersicina contains a type VI SQR enzyme (TrSqrF) having unusual catalytic parameters and four cysteines likely involved in the catalysis. Site-directed mutagenesis was applied to identify the role of cysteines in the catalytic process of TrSqrF. Based on biochemical and kinetic characterization of these TrSqrF variants, Cys121 is identified as crucial for enzyme activity. The cofactor is covalently bound via a heterodisulfide bridge between Cys121 and the C8M group of FAD. Mutation of another cysteine present in all SQRs (Cys332) causes remarkably decreased enzyme activity (14.6% of wild type enzyme) proving important, but non-essential role of this residue in enzyme catalysis. The sulfhydril-blocking agent, iodoacetamide can irreversibly inactivate TrSqrF but only if substrates are present and the enzyme is actively catalyzing its reaction. When the enzyme is inhibited by iodoacetamide, the FAD cofactor is released. The inhibition studies support a mechanism that entails opening and reforming of the heterodisulfide bridge during the catalytic cycle of TrSqrF. Our study thus reports the first detailed structure-function analysis of a type VI SQR enzyme which enables the proposal of a distinct mechanism of sulfide oxidation for this class.

Quinone binding site in a type VI sulfide:quinone oxidoreductase

“…S5). Likewise, previously published TrSqrF variants (Duzs et al 2021), a weak additional absorbance peak at 408 nm was observed in the UV-Vis spectra of these TrSqrF mutants, indicating the presence of trace amounts of c-type cytochromes in the samples (the extinction coefficient of heme-containing c-type cytochromes at 408 nm is high (170 mM −1 cm −1 ) (Sousa et al 2017;Sena et al 2018). The amount of c-type cytochrome contamination in SDS-PAGE is less than the detection limit (Fig.…”

“…The initial structure of the TrSqrF molecular dynamics (MD) calculations was the homology model previously described (Duzs et al 2021), in which the type VI SQR enzyme associated with dUQ and FAD cofactors was determined based on the crystal structures of A. ambivalens SqrE (Brito et al 2009) and A. aeolicus SqrA (Marcia et al 2010b). MD calculations were performed using the Desmond package (Schrödinger Release 2021-1) (Bowers et al 2006) of the Schrödinger software suites applying the OPLSA2005 (Jorgensen et al 1996) force field in combination with the SPC explicit water model.…”

“…The wild-type and a few StrepII affinity tag-fused TrSqrF variants were expressed in a T. roseopersicina strain (FOQRON) lacking sulfide oxidase enzymes (TrSqrD, TrSqrF, and FccAB) (Duzs et al 2018(Duzs et al , 2021. According to Duzs et al 2018, TrSqrF variants carrying mutations in the quinone binding pocket were generated.…”

“…The other Rossmann structure is primarily responsible for the catalytic site for sulfide binding and oxidation. In some enzymes, it also binds the cofactor via forming a covalent bond with the isoalloxazine ring of FAD (Duzs et al 2021). It has been reported that the C-terminal domain, which is composed of β-sheets and α-helical structural elements, plays a role in membrane binding and protein oligomerization (Lencina et al 2013(Lencina et al , 2020.…”

“…Additionally, this domain is responsible for hosting the binding site for the enzyme’s electron acceptor quinone substrates (Marcia et al 2009 , 2010a ; Cherney et al 2010 ; Zhang et al 2016 ). The C-terminal domain of most SQRs contains two amphipathic α-helices except for members of type VI group, which have a shorter C-terminal domain consisting of only one terminal α-helix (Duzs et al 2021 ) (Fig. 1 ).…”