Could a Diiron-Containing Four-Helix-Bundle Protein Have Been a Primitive Oxygen Reductase?

Gomes, Cláudio M.; Gall, Jean Le; Xavier, António V.; Teixeira, Miguel

doi:10.1002/1439-7633(20010803)2:7/8<583::aid-cbic583>3.0.co;2-5

Cited by 42 publications

(27 citation statements)

References 25 publications

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“…† No inhibition detected. enzymes that detoxified activated oxygen species by reducing them to H 2 O, and to have evolved instead to use diiron-bound activated oxygen species to effect catalysis (17). Evidence in support of this view came from the reversion of the evolved functionality of an SACPD back to that of an oxidase by a single residue substitution (18).…”

Section: Discussionmentioning

confidence: 99%

Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H ₂ O ₂ to the cosubstrate O ₂

André

Kim

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

118

121

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Biologically produced alkanes represent potential renewable alternatives to petroleum-derived chemicals. A cyanobacterial pathway consisting of acyl-Acyl Carrier Protein reductase and an aldehydedeformylating oxygenase (ADO) converts acyl-Acyl Carrier Proteins into corresponding n-1 alkanes via aldehyde intermediates in an oxygen-dependent manner (K m for O 2 , 84 ± 9 μM). In vitro, ADO turned over only three times, but addition of more ADO to exhausted assays resulted in additional product formation. While evaluating the peroxide shunt to drive ADO catalysis, we discovered that ADO is inhibited by hydrogen peroxide (H 2 O 2 ) with an apparent K i of 16 ± 6 μM and that H 2 O 2 inhibition is of mixed-type with respect to O 2 . Supplementing exhausted assays with catalase (CAT) restored ADO activity, demonstrating that inhibition was reversible and dependent on H 2 O 2 , which originated from poor coupling of reductant consumption with alkane formation. Kinetic analysis showed that long-chain (C14-C18) substrates follow Michaelis-Menten kinetics, whereas short and medium chains (C8-C12) exhibit substrate inhibition. A bifunctional protein comprising an N-terminal CAT coupled to a C-terminal ADO (CAT-ADO) prevents H 2 O 2 inhibition by converting it to the cosubstrate O 2 . Indeed, alkane production by the fusion protein is observed upon addition of H 2 O 2 to an anaerobic reaction mix. In assays, CAT-ADO turns over 225 times versus three times for the native ADO, and its expression in Escherichia coli increases catalytic turnovers per active site by fivefold relative to the expression of native ADO. We propose the term "protection via inhibitor metabolism" for fusion proteins designed to metabolize inhibitors into noninhibitory compounds.adlehyde decarbonylase | diiron enzyme | dinuclear iron | enzyme regulation

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Section: Discussionmentioning

confidence: 99%

Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H ₂ O ₂ to the cosubstrate O ₂

André

Kim

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

118

121

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“…Our results are the first to suggest such a role, which is not without precedence in oxidase activity (40). In this respect, it is very interesting to note that a conserved tyrosine is also present (at a locus similar to that of Tyr-275 in the AOX) in rubrerythrin, a di-iron carboxylate protein that has recently been shown to exhibit oxidase activity (41). Clearly, further studies are required to elucidate the exact role of Tyr-275 and other conserved residues in the catalytic mechanism of the AOX.…”

Section: Discussionmentioning

confidence: 99%

Structure of the Plant Alternative Oxidase

Albury

Affourtit

Crichton

et al. 2002

Journal of Biological Chemistry

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All higher plants and many fungi contain an alternative oxidase (AOX), which branches from the cytochrome pathway at the level of the quinone pool. In an attempt, first, to distinguish between two proposed structural models of this di-iron protein, and, second, to examine the roles of two highly conserved tyrosine residues, we have expressed an array of site-specific mutants in Schizosaccharomyces pombe. Mitochondrial respiratory analysis reveals that S. pombe cells expressing AOX proteins in which Glu-217 or Glu-270 were mutated, no longer exhibit antimycin-resistant oxygen uptake, indicating that these residues are essential for AOX activity. Although such data corroborate a model that describes the AOX as an interfacial membrane protein, they are not in full agreement with the most recently proposed ligation sphere of its di-iron center. We furthermore show that upon mutation of Tyr-253 and Tyr-275 to phenylalanines, AOX activity is fully maintained or abolished, respectively. These data are discussed in reference to the importance of both residues in the catalytic cycle of the AOX. The alternative oxidase (AOX)1 is a ubiquinol:oxygen oxidoreductase that catalyzes the four electron reduction of oxygen to water (for recent reviews, see Refs. 1-4). This terminal oxidase branches from the cytochrome pathway at the level of the ubiquinone pool and is non-protonmotive. The AOX is resistant to inhibitors of the cytochrome pathway such as cyanide and antimycin A but inhibited by a number of compounds including hydroxamic acids (e.g. salicylhydroxamic acid) and n-alkyl-gallates.Two structural models of the AOX currently exist. The first model proposed by Siedow et al. (5,6) was based on relatively few AOX sequences and classified the AOX as a member of the di-iron family of proteins that also includes the R2 subunit of ribonucleotide reductase and the hydroxylase component of methane monooxygenase. Based on hydropathy analysis, the AOX was predicted to contain two transmembrane helices that are connected by a helix located in the intermembrane space (6) (Fig. 1A). Since this model was proposed, further AOX sequences were identified, resulting in the proposal by Andersson and Nordlund (7) of an alternative structural model (Fig. 1B). Although this second model also classifies the AOX as a di-iron protein, it differs in the precise ligation sphere of the di-iron center (7). For instance, one of the C-terminal Glu-X-X-His motifs identified by Siedow et al. (5,6), containing Glu-270, appeared not to be fully conserved in the newly identified sequences and consequently seemed unlikely to play a role in ligating iron. Instead, Andersson and Nordlund used a third Glu-X-X-His motif (that contains Glu-217, which is located in the intermembrane space according to the Siedow et al. model) to coordinate the iron atoms. Since such a choice implies that the transmembrane helices can no longer be retained, Andersson and Nordlund (7) proposed that the AOX is an interfacial rather than a transmembrane protein.Recently, the IMMUTANS (...

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“…Oxygen activation likely placed strong evolutionary constraints on the organization of the diiron center, whereas the reaction phases exhibit great diversity of functional outcome. In addition to their individual catalytic reactions, rubrerythrin, methane monooxygenase, ribonucleotide reductase, and the ⌬ 9 desaturase have also been shown to reduce dioxygen to water (4)(5)(6). Based on these similarities, Gomes et al (4) proposed that the four-helix bundle diiron proteins arose from a common ancestor that bound activated oxygen species and reduced them to water.…”

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confidence: 85%

A single mutation in the castor Δ ⁹ -18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry

Guy

Abreu²,

Moche³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Sequence analysis of the diiron cluster-containing soluble desaturases suggests they are unrelated to other diiron enzymes; however, structural alignment of the core four-helix bundle of desaturases to other diiron enzymes reveals a conserved iron binding motif with similar spacing in all enzymes of this structural class, implying a common evolutionary ancestry. Detailed structural comparison of the castor desaturase with that of a peroxidase, rubrerythrin, shows remarkable conservation of both identity and geometry of residues surrounding the diiron center, with the exception of residue 199. Position 199 is occupied by a threonine in the castor desaturase, but the equivalent position in rubrerythrin contains a glutamic acid. We previously hypothesized that a carboxylate in this location facilitates oxidase chemistry in rubrerythrin by the close apposition of a residue capable of facilitating proton transfer to the activated oxygen (in a hydrophobic cavity adjacent to the diiron center based on the crystal structure of the oxygen-binding mimic azide). Here we report that desaturase mutant T199D binds substrate but its desaturase activity decreases by Ϸ2 ؋ 10 3 -fold. However, it shows a >31-fold increase in peroxide-dependent oxidase activity with respect to WT desaturase, as monitored by single-turnover stopped-flow spectrometry. A 2.65-Å crystal structure of T199D reveals active-site geometry remarkably similar to that of rubrerythrin, consistent with its enhanced function as an oxidase enzyme. That a single amino acid substitution can switch reactivity from desaturation to oxidation provides experimental support for the hypothesis that the desaturase evolved from an ancestral oxidase enzyme.binuclear ͉ diiron ͉ enzyme N onheme diiron-containing four-helix-bundle proteins possess the ability to functionalize unactivated C-H groups and mediate a diversity of chemical reactions including oxidation, hydroxylation, desaturation, and epoxidation (1, 2). A wealth of mechanistic information is available from various diironcontaining proteins including methane monooxygenases, ⌬ 9 desaturases, ribonucleotide reductases, rubrerythrins, alternate oxidases, ferritins, and bacterioferritins (1-3).The diiron-containing proteins are highly divergent in their amino acid sequences, with identities typically falling below that necessary for conventional phylogenetic analysis. However, when the analysis is restricted to the four helices that coordinate the diiron active site, the amino acid identity rises to 16-31% (4). A shared diiron-binding motif within the conserved four-helix bundle is involved in oxygen chemistry. The reactions have been described as occurring in two phases, an oxygen activation phase followed by reaction phases (1). Oxygen activation likely placed strong evolutionary constraints on the organization of the diiron center, whereas the reaction phases exhibit great diversity of functional outcome. In addition to their individual catalytic reactions, rubrerythrin, methane monooxygenase, ribonucleotide reductas...

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Could a Diiron-Containing Four-Helix-Bundle Protein Have Been a Primitive Oxygen Reductase?

Cited by 42 publications

References 25 publications

Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H ₂ O ₂ to the cosubstrate O ₂

Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H ₂ O ₂ to the cosubstrate O ₂

Structure of the Plant Alternative Oxidase

A single mutation in the castor Δ ⁹ -18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry

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Could a Diiron-Containing Four-Helix-Bundle Protein Have Been a Primitive Oxygen Reductase?

Cited by 42 publications

References 25 publications

Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H 2 O 2 to the cosubstrate O 2

Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H 2 O 2 to the cosubstrate O 2

Structure of the Plant Alternative Oxidase

A single mutation in the castor Δ 9 -18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry

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Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H ₂ O ₂ to the cosubstrate O ₂

Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H ₂ O ₂ to the cosubstrate O ₂

A single mutation in the castor Δ ⁹ -18:0-desaturase changes reaction partitioning from desaturation to oxidase chemistry