No abstract

Sulfide:quinone oxidoreductases (SQRs) are ubiquitous enzymes which have multiple roles: sulfide detoxification, energy generation by providing electrons to respiratory or photosynthetic electron transfer chains, and sulfide homeostasis. A recent structure-based classification defines 6 groups of putative SQRs (I – VI), and representatives of all but group III have been confirmed to have sulfide oxidase activity. In the current work, we report the first characterization of a predicted group III SQR from Caldivirga maquilingensis, and confirm that this protein is a sulfide oxidase. The gene encoding the enzyme was cloned, and the protein was expressed in E. coli and purified. The enzyme oxidizes sulfide using decylubiquinone as an electron acceptor, and is inhibited by aurachin C and iodoacetamide. Analysis of the amino acid sequence indicates that the C. maquilingensis SQR has two amphiphilic helices at the C-terminus but lacks any transmembrane helices. This suggests that C. maquilingensis SQR interacts with the membrane surface and that the interactions are mediated by the C-terminal amphiphilic helices. Mutations within the last C-terminal amphiphilic helix resulted in a water-soluble form of the enzyme which, remarkably, retains full SQR activity using decylubiquinone as the electron acceptor. Mutations at one position, L379, also located in the C-terminal amphiphilic helix, inactivated the enzyme by preventing the interaction with decylubiquinone. It is concluded that the C-terminal amphiphilic helix is important for membrane binding and for forming part of the pathway providing access of the quinone substrate to the protein-bound flavin at the enzyme active site.

Cytochrome bo is a respiratory proton-pumping oxygen reductase that is a member of the heme-copper superfamily that utilizes ubiquinol-8 (QH) as a substrate. The current consensus model has QH oxidized at a low affinity site (Q), passing electrons to a tightly bound quinone cofactor at a high affinity site (Q site) that stabilizes the one-electron reduced ubisemiquinone, facilitating the transfer of electrons to the redox active metal centers where O is reduced to water. The current work shows that the Q bound to the Q site is more dynamic than previously thought. In addition, mutations of residues at the Q site that do not abolish activity have been re-examined and shown to have properties expected of mutations at the substrate binding site (Q): an increase in the K of the substrate ubiquinol-1 (up to 4-fold) and an increase in the apparent K of the inhibitor HQNO (up to 8-fold). The data suggest that there is only one binding site for ubiquinol in cyt bo and that site corresponds to the Q site.

Significance Quinol oxidases that are members of the heme–copper superfamily of respiratory oxygen reductases have evolved from cytochrome c oxidases. They directly oxidize quinol and reduce oxygen to water. Here, we describe two high-resolution cryogenic electron microscopy structures of the proton-pumping cytochrome bo 3 ubiquinol oxidase in styrene–maleic acid copolymer nanodiscs and in membrane scaffold protein nanodiscs. Each structure contains one equivalent of well-resolved ubiquinone-8 in the substrate binding site as well as several phospholipid molecules. These structures indicate that H98 I has two conformations that allow H98 I hydrogen bonded to carbonyl O4 of the UQ8 or with E14 I . We propose that H98 I dynamics serves to shuttle protons from ubiquinol-8 via E14 I to the bulk aqueous phase upon ubiquinol-8 oxidation.

Breaking down oxygen Molecular oxygen (O 2 ) is the terminal oxidant for respiration in mitochondria and many bacteria. Within membrane-bound heme–copper oxidases, a controlled, four-electron reduction of O 2 to water is coupled to pumping of protons across the membrane that can be used, among other outcomes, to generate adenosine triphosphate. Studying cytochrome bo 3 ubiquinol oxidase, Jose et al . investigated the key P M intermediate, which forms after O–O bond cleavage and precedes proton pumping, using magnetic circular dichroism spectroscopy. The authors observed features demonstrating that P M is a three-spin system, which is consistent with a consensus model including an iron(IV)-oxo species, copper(II) ion, and tyrosyl radical. These results provide an important validation of the O–O cleavage mechanism and open the door to understanding the proton pumping step. —MAF