Functional characterization of CYP4G11-a highly conserved enzyme in the western honey bee Apis mellifera.

Determining the functionality of CYP4G11, the only CYP4G in the genome of the western honey bee Apis mellifera, can provide insight into its reduced CYP4 inventory. Toward this objective, CYP4G11 transcripts were quantified, and CYP4G11 was expressed as a fusion protein with housefly CPR in Sf9 cells. Transcript levels varied with age, task, and tissue type in a manner consistent with the need for cuticular hydrocarbon production to prevent desiccation or with comb wax production. Young larvae, with minimal need for desiccation protection, expressed CYP4G11 at very low levels. Higher levels were observed in nurses, and even higher levels in wax producers and foragers, the latter of which risk desiccation upon leaving the hive. Recombinant CYP4G11 readily converted octadecanal to n-heptadecane in a time-dependent manner, demonstrating its functions as an oxidative decarbonylase. CYP4G11 expression levels are high in antennae; heterologously expressed CYP4G11 converted tetradecanal to n-tridecane, demonstrating that it metabolizes shorter-chain aldehydes. Together, these findings confirm the involvement of CYP4G11 in cuticular hydrocarbon production and suggest a possible role in clearing pheromonal and phytochemical compounds from antennae. This possible dual functionality of CYP4G11, i.e., cuticular hydrocarbon and comb wax production and antennal odorant clearance, may explain how honey bees function with a reduced CYP4G inventory.

Comparative transcriptomics of mountain pine beetle pheromone-biosynthetic tissues and functional analysis of CYP6DE3.

BACKGROUND: The mountain pine beetle (MPB, Dendroctonus ponderosae Hopkins) is a highly destructive pest of pine forests in western North America. During flight to a new host tree and initiation of feeding, mountain pine beetles release aggregation pheromones. The biosynthetic pathways of these pheromones are sex-specific and localized in the midgut and fat body, but the enzymes involved have not all been identified or characterized. RESULTS: We used a comparative RNA-Seq analysis between fed and unfed male and female MPB midguts and fat bodies to identify candidate genes involved in pheromone biosynthesis. The 13,407 potentially unique transcripts showed clear separation based on feeding state and gender. Gene co-expression network construction and examination using petal identified gene groups that were tightly connected. This, as well as other co-expression and gene ontology analyses, identified all four known pheromone biosynthetic genes, confirmed the tentative identification of four others from a previous study, and suggested nine novel candidates. One cytochrome P450 monooxygenase, CYP6DE3, identified as a possible exo-brevicomin-biosynthetic enzyme in this study, was functionally characterized and likely is involved in resin detoxification rather than pheromone biosynthesis. CONCLUSIONS: Our analysis supported previously characterized pheromone-biosynthetic genes involved in exo-brevicomin and frontalin biosynthesis and identified a number of candidate cytochrome P450 monooxygenases and a putative cyclase for further studies. Functional analyses of CYP6DE3 suggest its role in resin detoxification and underscore the limitation of using high-throughput data to tentatively identify candidate genes. Further functional analyses of candidate genes found in this study should lead to the full characterization of MPB pheromone biosynthetic pathways and the identification of molecular targets for possible pest management strategies.

Isolation, endocrine regulation and transcript distribution of a putative primary JH-responsive gene from the pine engraver, Ips pini (Coleoptera: Scolytidae).

We isolated a cDNA of unknown function from a juvenile hormone III (JH III)-treated male midgut cDNA library prepared from the pine engraver beetle, Ips pini, and examined its genomic structure. The gene, tentatively named "Ipi10G08", encoded a 410 amino acid translation product that shared 26-37% identity with unannotated matches from several insects. Semi-quantitative RT-PCR analysis of Ipi10G08 following application of a 10 microg dose of JH III demonstrated an early induction for both male and female beetles, with transcripts being detectable after 45 min. An expression profile of male midgut tissue indicated Ipi10G08 transcript levels reach a maximum induction of approximately 22.5-fold control levels at 4h post-treatment. Tissue distribution studies displayed a large induction of Ipi10G08 mRNA in the alimentary canal of JH III-treated beetles, especially in males. A dose curve from both sexes suggested there may be a difference in the ability to respond to lower levels of JH III and immunoblot analysis indicated that although JH III highly induces transcript levels in females, protein levels are not similarly induced, while protein levels are induced in males. Ipi10G08 is likely a primary JH response gene and may provide insight into how this hormone exerts its actions.

Isolation, endocrine regulation and mRNA distribution of the 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-S) gene from the pine engraver, Ips pini (Coleoptera: Scolytidae).

We isolated a full-length cDNA encoding 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-S) from the pine engraver beetle, Ips pini (Say), and examined its genomic structure. The intron-less gene has a predicted 460 amino acid cytosolic protein product with 73% identity to HMG-S from Dendroctonus jeffreyi, and high identity (58-64%) with other insect HMG-Ss. Topically applied juvenile hormone (JH) III induced HMG-S mRNA levels up to 6.5-fold in both sexes, mostly in the anterior midgut, though there were differences between males and females in the timing, sensitivity to JH III dose and tissue distribution of HMG-S mRNA. These data further validate the coordinate regulation of mevalonate pathway genes for de novo isoprenoid pheromone production in bark beetles.

Effects of juvenile hormone on gene expression in the pheromone-producing midgut of the pine engraver beetle, Ips pini.

Juvenile hormone III (JH III) stimulates biosynthesis of the monoterpenoid aggregation pheromone component, ipsdienol, in the anterior midgut of the male pine engraver beetle, Ips pini (Say). To understand better the hormonal regulation of pheromone biosynthesis in this forest pest, and identify JH III-responsive genes, microarrays were prepared and hybridized to cDNA from midguts of JH III-treated beetles. Expression patterns were confirmed by quantitative real-time RT-PCR. JH III co-ordinately regulated mevalonate pathway genes and many other genes implicated in pheromone biosynthesis. Sex differences in basal levels of mevalonate pathway genes were consistent with their role in male-specific pheromone biosynthesis. This is the first microarray-based study of the developmental and hormonal regulation of insect pheromone biosynthesis.

Isolation and molecular characterization of Musca domestica delta-9 desaturase sequences.

We have isolated fatty acyl-CoA desaturase cDNA (Mdomd9) and genomic sequences from the housefly, Musca domestica. Two approximately 1.66 kb cDNAs were recovered. They had identical coding regions and 3' untranslated regions (UTRs), but differed in their 5' UTRs. The open reading frame encodes a 380 amino acid (aa) protein with 82% identity to Drosophila melanogaster desat1, and significant (> 50%) identity with other insect delta-9 desaturases. Functional analyses in a yeast expression system confirmed the cDNA encodes a delta9 desaturase. Northern analysis indicated two transcripts of 1.7 and 2.9 kb that hybridized specifically to the open reading frame. PCR amplification of genomic templates revealed three intron sites that are conserved among other insect species. Southern analysis of genomic DNA indicated at least two desaturase gene copies per haploid genome. There is a high degree of polymorphism, most of which appears to be due to variable intron sequences; curiously, individual flies had varying morphs of intron II and intron III. Together, the data suggest that there are more delta9 desaturase alleles within the population studied than there are loci within the genome, and support other studies suggesting that insect fatty acyl-CoA desaturases are a dynamically evolving gene family.

Biochemical and molecular characterizaton of house cricket (Acheta domesticus, Orthoptera: Gryllidae) Delta9 desaturase.

Studies of insect fatty acyl-CoA desaturases have heretofore concentrated on the Diptera and Lepidoptera. We report here the isolation and characterization of a fatty acyl-CoA Delta9 desaturase from the house cricket, Acheta domesticus (Orthoptera). Two desaturase cDNAs were isolated from a library, one of which contained two intron sequences. The clones were identical in their respective coding regions, but had divergent 5' and 3' untranslated regions. The cDNAs encode a 359 amino acid desaturase enzyme that could rescue a fatty acyl-CoA desaturase auxotrophic phenotype when expressed in yeast. Biochemical analysis of lipids from transformed yeast cells confirmed that the enzyme is a Delta9 desaturase with activity on both palmitic and stearic acid substrates. Southern blotting indicated multiple Delta9 desaturase genes within the genome. A single message that was up-regulated in fed insects vs. starved insects was observed on northern blots, indicating transcriptional regulation in response to diet.

Specialization of midgut cells for synthesis of male isoprenoid pheromone components in two scolytid beetles, Dendroctonus jeffreyi and Ips pini.

Endodermal or midgut cells have only recently been recognized as the site of pheromone synthesis in bark beetles. Midgut cells are not only specialized for digestion, but they have also been recruited to form isoprenoid compounds that function as pheromone components in Ips pini and Dendroctonus jeffreyi. Male bark beetle midgut cells are competent to produce isoprenoid pheromones after feeding or stimulation by juvenile hormone (JH) III. Competent midgut cells share many ultrastructural features with cells that do not secrete isoprenoid pheromone, but they are distinguished from these by abundant and highly ordered arrays of smooth endoplasmic reticula. During secretion, both midgut cells that produce pheromone and cells that do not are characterized by the presence of apical extrusions (apocrine secretion) rather than the presence of vesicles that fuse with the apical membrane and undergo exocytosis (eccrine secretion). Pheromone-producing cells of the midgut do not represent a population of cells that are distinct from cells involved in digestion. All, or most, midgut cells of male I. pini and D. jeffreyi can secrete pheromones as well as digestive enzymes.

Male jeffrey pine beetle, Dendroctonus jeffreyi, synthesizes the pheromone component frontalin in anterior midgut tissue.

The male Jeffrey pine beetle, Dendroctonus jeffreyi Hopkins (Coleoptera: Scolytidae), produces the bicyclic ketal frontalin as part of a complex semiochemical blend. A key regulated enzyme in the mevalonate pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R), showed high transcript levels in the anterior midgut of male Jeffrey pine beetles by in situ hybridization. HMG-R expression in this area of the alimentary canal was related to male emergence, where emerged males demonstrated significant up-regulation of HMG-R transcript and pre-emerged males showed only basal levels. Pre-emerged males were induced to express high levels of HMG-R transcript by treatment with juvenile hormone (JH) III. Additionally, isolated anterior midgut tissue from JH III-treated males converted radiolabeled acetate to frontalin, as assayed by radio-HPLC, providing strong evidence that this is the site of frontalin production in male beetles.

Hydrocarbons of Rhodnius prolixus, a Chagas disease vector.

The surface hydrocarbons of the blood-sucking insect, Rhodnius prolixus, a major Chagas disease vector in Venezuela, Colombia and Central America, were characterized by capillary gas chromatography coupled to mass spectrometry (CGC-MS). A total of 54 single or multicomponent peaks of saturated, straight-chain and methyl-branched hydrocarbons were identified. Major n-alkanes were n-C27, n-C29, n-C31 and n-C33 hydrocarbons. In the branched fraction, methyl groups were at positions 3, 5, 7, 11, 13, 15 and 17- for monomethyl isomers, and separated by three or five methylene groups for the trimethyl or tetramethyl derivatives. For the higher molecular weight components of 37, 39 and 41 atoms in the carbon skeleton, the di-, tri- and tetramethyl branches were usually separated by three or five, and sometimes 7, 11 or 13, methylene groups. The internal hydrocarbon pool contained larger amounts of the higher molecular weight methyl-branched components. Qualitative differences among epicuticular and internal hydrocarbon compositions were detected, both in adult and nymphal stages. No significant sexual dimorphism was detected, but a significant shift in the major n-alkane components was evident from the nymphal to the adult stage, differing also in the relative amounts of the higher molecular weight methyl-branched chains. Comparison of the hydrocarbon components to that of other Chagas disease vectors is discussed.

Hydrocarbon-released nestmate aggression in the Argentine ant, Linepithema humile, following encounters with insect prey.

Argentine ants, Linepithema humile, were attacked by their nestmates following contact with a particular prey item, the brown-banded cockroach, Supella longipalpa. Contact with prey, as brief as 2 min, provoked nestmate aggression. Argentine ants contaminated with hydrocarbons extracted from S. longipalpa also released nestmate aggression behavior similar to that released by the whole prey item, confirming the involvement of hydrocarbons. In contrast to S. longipalpa, little or no nestmate aggression was induced by other ant prey from diverse taxa. A comparison of prey hydrocarbon profiles revealed that all hydrocarbons of S. longipalpa were very long chain components with 33 or more carbons, while other prey had either less, or none, of the very long chain hydrocarbons of 33 carbons or greater. We identified the hydrocarbons of S. longipalpa and some new groups of long chain hydrocarbons of L. humile. The majority of S. longipalpa hydrocarbons were 35 and 37 carbons in length with one to three methyl branches, and closely resembled two previously unidentified groups of compounds from L. humile of similar chain length. The hydrocarbons of S. longipalpa and L. humile were compared and their role in the Argentine ant nestmate recognition is discussed.

Effect of age and sex on the production of internal and external hydrocarbons and pheromones in the housefly, Musca domestica.

The epicuticular and internal waxes of male and female houseflies were examined by capillary gas chromatography-mass spectrometry at closely timed intervals from emergence until day-6 of adulthood. New components identified included tricosan-10-one, 9,10-epoxyheptacosane, heptacosen-12-one, a series of odd-carbon numbered dienes from C31 to C39, several positional isomers of monoenes including (Z)-9- and 7-pentacosene and a number of methyl- and dimethylalkanes. (Z)-9-tricosene appears in internal lipids prior to appearing on the surface of the insect, suggesting that it is transported in the hemolymph to its site of deposition on the epicuticle. The large increases in the amount of (Z)-9-tricosene in females from day-2 until day-6 is compensated for by a concomitant decrease in (Z)-9-heptacosene. The C23 epoxide and ketone only appear in females after the production of (Z)-9-tricosene is induced, and are only abundant in epicuticular waxes, suggesting they are formed after (Z)-9-tricosene is transported to the cells which are involved in taking them to the surface of the insect. Mathematical analysis indicated that the time shift between internal production and external accumulation in females is more than 24 h. The divergence between male and female lipid production occurs at an early stage, when insects are less than one day old.

Tissue distribution and lipophorin transport of hydrocarbons and sex pheromones in the house fly, Musca domestica.

We investigated the relationship between epicuticular and internal hydrocarbons in the adult house fly, Musca domestica and the distribution of hydrocarbons, including the female sex pheromone component, (Z)-9-tricosene, in tissues. Internal hydrocarbons increased dramatically in relation to sexual maturation and were found in the hemolymph, ovaries, digestive tract, and fat body. (Z)-9-Tricosene comprised a relatively large fraction of the hydrocarbons in the female carcass and hemolymph, and less so in other tissues, while other hydrocarbons were represented in greater amounts in the ovaries than in other tissues. It therefore appears that certain hydrocarbons were selectively provisioned to certain tissues such as the ovaries, from which pheromone was relatively excluded. Both KBr gradient ultracentrifugation and specific immunoprecipitation indicated that > 90% of hemolymph hydrocarbons were associated with a high-density lipophorin (density = 1.09 g ml(-1)), composed of two apoproteins under denaturing conditions, apolipophorin I (approximately 240 kD) and apolipophorin II (approximately 85 kD). Our results support a predicted model (Chino, 1985) that lipophorin is involved in the transport of sex pheromone in M. domestica. In addition to delivering hydrocarbons and sex pheromones to the cuticular surface, we suggest that lipophorin may play an important role in an active mechanism that selectively deposits certain subsets of hydrocarbons at specific tissues.

Isolation and endocrine regulation of an HMG-CoA synthase cDNA from the male Jeffrey pine beetle, Dendroctonus jeffreyi (Coleoptera: Scolytidae).

We have isolated a full length 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-S) cDNA from the male Jeffrey pine beetle, Dendroctonus jeffreyi Hopkins, and studied the effects of topical applications of juvenile hormone III (JH III) on its expression. The predicted translation product of this apparently single copy gene has 63% and 58% identity with HMG-S1 and HMG-S2 from Blattella germanica (L.), and 61% identity with Drosophila melanogaster Hmgs. HMG-S transcript levels remain uniformly low in JH III-treated and control D. jeffreyi females, but are induced approximately 2.5- to 5-fold in JH III-treated males. JH III causes a dose- and time-dependent increase in HMG-S transcripts in the male metathoracic-abdominal region. Since monoterpenoid pheromone precursor synthesis and HMG-CoA reductase expression are under the control of JH III in the metathorax of Ips bark beetles, the observed HMG-S expression pattern suggests that the isoprenoid pathway is similarly important for semiochemical production in D. jeffreyi.

Effects of developmental age, ambient temperature, and dietary alterations on delta(12) desaturase activity in the house cricket, Acheta domesticus.

Double bond formation in polyunsaturated fatty acids (PUFA) is mediated by desaturase enzymes. Certain insect species have been found to possess a Delta(12) desaturase, previously thought to occur exclusively in plants. We have begun to characterize this enzyme to determine its relatedness to those found in plants and animals. Desaturase activity can be altered significantly by a number of environmental factors in protozoa, cyanobacteria, plants, fish, and rats. We present evidence here that Delta(12) desaturase activity in Acheta domesticus is affected by developmental stage, starvation, dietary alterations, and fluctuations in ambient temperature. Highest activity is observed during the middle of the penultimate instar and 3 to 6 days after adult emergence. Starvation markedly decreases Delta(12) activity, whereas resumption of feeding on fat-free or low fat diets increases activity.

Insect pheromones--an overview of biosynthesis and endocrine regulation.

This overview describes, compares, and attempts to unify major themes related to the biosynthetic pathways and endocrine regulation of insect pheromone production. Rather than developing and dedicating an entirely unique set of enzymes for pheromone biosynthesis, insects appear to have evolved to add one or a few tissue-specific auxiliary or modified enzymes that transform the products of "normal" metabolism to pheromone compounds of high stereochemical and quantitative specificity. This general understanding is derived from research on model species from one exopterygote insect order (Blattodea) and three endopterygote insect orders (Coleoptera, Diptera, and Lepidoptera). For instance, the ketone hydrocarbon contact sex pheromone of the female German cockroach, Blattella germanica, derives its origins from fatty acid biosynthesis, arising from elongation of a methyl-branched fatty acyl-CoA moiety followed by decarboxylation, hydroxylation, and oxidation. Coleopteran sex and aggregation pheromones also arise from modifications of fatty acid biosynthesis or other biosynthetic pathways, such as the isoprenoid pathway (e.g. Cucujidae, Curculionidae, and Scolytidae), or from simple transformations of amino acids or other highly elaborated host precursors (e.g. Scarabaeidae and Scolytidae). Like the sex pheromone of B. germanica, female-produced dipteran (e.g. Drosophilidae and Muscidae) sex pheromone components originate from elongation of fatty acyl-CoA moieties followed by loss of the carbonyl carbon and the formation of the corresponding hydrocarbon. Female-produced lepidopteran sex pheromones are also derived from fatty acids, but many moths utilize a species-specific combination of desaturation and chain-shortening reactions followed by reductive modification of the carbonyl carbon. Carbon skeletons derived from amino acids can also be used as chain initiating units and elongated to lepidopteran pheromones by this pathway (e.g. Arctiidae and Noctuidae). Insects utilize at least three hormonal messengers to regulate pheromone biosynthesis. Blattodean and coleopteran pheromone production is induced by juvenile hormone III (JH III). In the female common house fly, Musca domestica, and possibly other species of Diptera, it appears that during hydrocarbon sex pheromone biosynthesis, ovarian-produced ecdysteroids regulate synthesis by affecting the activities of one or more fatty acyl-CoA elongation enzyme(s) (elongases). Lepidopteran sex pheromone biosynthesis is often mediated by a 33 or 34 amino acid pheromone biosynthesis activating neuropeptide (PBAN) through alteration of enzyme activities at one or more steps prior to or during fatty acid synthesis or during modification of the carbonyl group. Although a molecular level understanding of the regulation of insect pheromone biosynthesis is in its infancy, in the male California fivespined ips, Ips paraconfusus (Coleoptera: Scolytidae), JH III acts at the transcriptional level by increasing the abundance of mRNA for 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme in de novo isoprenoid aggregation pheromone biosynthesis.

Juvenile hormone regulation of HMG-R gene expression in the bark beetle Ips paraconfusus (Coleoptera: Scolytidae): implications for male aggregation pheromone biosynthesis.

Juvenile hormone III (JH III) induces acyclic isoprenoid pheromone production in male Ips paraconfusus. A likely regulatory enzyme in this process is 3-hydroxy-e-methylglutaryl-CoA reductase (HMG-R). To begin molecular studies on pheromone production, a 1.16-kb complementary DNA representing approximately one-third of I. paraconfusus HMG-R was isolated by polymerase chain reaction and sequenced. The predicted translation product is 59% and 75% identical to the corresponding portion of HMG-R from the fruit fly, Drosophila melanogaster, and the German cockroach, Blattella germanica, respectively. Northern blots show that topical application of JH III increases HMG-R transcript levels in male thoraces in an apparent dose- and time-dependent manner. These data support the model that JH III raises HMG-R transcript levels, resulting in increased activity of the isoprenoid pathway and de novo pheromone production.

Cuticular hydrocarbons and their role in copulatory behavior in Phormia regina (Meigen).

The cuticular hydrocarbons from adult Phormia regina (Meigen) were characterized by gas chromatography-mass spectrometry. Both sexes had similar components in nearly identical quantities, consisting of complex mixtures of saturated n-, monomethyl- and dimethylalkanes from 23 to 33 total carbons. Although no diet-, age-, or sex-specific differences were observed, cuticular hydrocarbons were shown to be involved in copulatory behavior. Hydrocarbon profiles of wild, compared to laboratory reared flies, showed no major differences. Behaviorally, males responded the same to dead decoys of either sex. Removal of the hydrocarbons, using hexane, from either male or female decoys, did not affect the number of mating strikes, but markedly reduced the number of copulatory attempts and the amount of time males spent mounted on either decoy. House fly, Musca domestica L., males when paired with a female M. domestica decoy produced copulatory attempts: whereas, when P. regina males were placed with M. domestica female decoys, there were no copulatory attempts. It is concluded that the cuticular hydrocarbons of P. regina function as species-specific but not sex-specific mating cues and elicit species-specific copulatory behavior in males.

Characterization of a novel microsomal fatty acid synthetase (FAS) compared to a cytosolic FAS in the housefly, Musca domestica.

A novel membrane-bound fatty acid synthetase (FAS) associated with the microsomal fraction from the housefly, Musca domestica, was solubilized and purified to homogeneity. The microsomal FAS was solubilized by 0.75 M KCl in phosphate buffer and was purified to homogeneity by the sequential use of ammonium sulfate precipitation followed by Sepharose CL-6B, DEAE Sephacel and Red Agarose (dye ligand affinity) chromatography. The specific activity of the microsomal FAS was increased 1,440-fold to 6,522 U/mg during purification. The cytosolic FAS from the housefly was also purified by similar methods and the specific activity increased 183-fold to 7,533 U/mg. The relative molecular mass of the microsomal and cytosolic FAS are 419 +/- 22 kDa and 405 +/- 18 kDa, respectively, for the dimers as determined by gel permeation chromatography. The microsomal and the cytosolic FAS yield different tryptic digestion maps and have slightly different amino acid compositions, which demonstrate structural differences between the two FASs. In addition, there are differences between the two FASs in their kinetic characteristics and their ability to incorporate methylmalonylCoA into the growing fatty acyl chain.

Proposed mechanism for the cytochrome P450-catalyzed conversion of aldehydes to hydrocarbons in the house fly, Musca domestica.

Experiments were performed to elucidate the mechanism of hydrocarbon formation in microsomal preparations from the house fly, Musca domestica. Antibody to both house fly cytochrome P450 reductase and a purified cytochrome P450 (CYP6A1) from the house fly inhibited (Z)-9-tricosene (Z9-23:Hy) formation from [15,16-3H]-(Z)-15-tetracosenal (24:1 aldehyde). Chemical ionization-gas chromatography-mass spectrometry (CI-GC-MS) analyses of the n-tricosane formed by microsomal preparations from [2,2-2H2,2-13C]- and [3,3-2H2,3-13C]tetracosanoyl-CoA demonstrated that the deuteriums on the 2,2- and 3,3-positions were retained in the conversion to the hydrocarbon product. Likewise, CI-GC-MS analysis of the Z9-23:Hy formed from [1-2H]tetracosenal by microsomal preparations demonstrated that the aldehydic proton on the 1-carbon was transferred to the hydrocarbon product. Hydrogen peroxide, cumene hydroperoxide, and iodosobenzene were able to support hydrocarbon production from [3H]24:1 aldehyde in place of O2 and NADPH for short incubation times. From these data, a cytochrome P450 mechanism is proposed in which the perferryl iron-oxene, resulting from heterolytic cleavage of the O-O bond of the iron-peroxy intermediate, abstracts an electron from the C=O double bond of the carbonyl group of the aldehyde. The reduced perferryl attacks the 1-carbon of the aldehyde to form a thiyl-iron-hemiacetal diradical. The latter intermediate can fragment to form an alkyl radical and a thiyl-iron-formyl radical. The alkyl radical then abstracts the formyl hydrogen to produce the hydrocarbon and CO2.