Sterol Side Chain Research Articles

Analysis of the mechanisms regulating the expression of isoprenoid biosynthesis genes in hydroponically-grown Nicotiana benthamiana plants using virus-induced gene silencing

Secondary metabolites in plants play important roles in defence against biotic and abiotic stresses. Although the biosynthesis pathways of secondary metabolites have been extensively studied, the regulatory mechanism of gene expression involved in these pathways remains poorly understood. In this study, we develop a virus-induced gene silencing (VIGS) system that enables a rapid analysis of the regulatory mechanism of genes involved in the biosynthesis of isoprenoids, one of the largest groups in secondary metabolites, using hydroponically-grown Nicotiana benthamiana. Using VIGS, we successfully reduced the transcript levels of 3-hydroxy-3-methylglutaryl-CoA reductase 1 (HMGR1), cycloartenol synthase 1 (CAS1), sterol side chain reductase 2 (SSR2) and S-adenosyl-L-Met-dependent C-24 sterol methyltransferase 1 (SMT1) in leaf, stem and root tissues in approximately 2 weeks. We identified novel feedback and feed-forward regulation of isoprenoid biosynthesis genes when CAS1, which encodes a key enzyme involved in the biosynthesis of sterols and steroidal glycoalkaloids, was down-regulated. Furthermore, the regulation of these genes differed among different tissues. These results demonstrate that our system can rapidly analyse the regulatory mechanisms involved in the biosynthesis of secondary metabolites.

Overexpression of cytochrome p450 125 in Mycobacterium: a rational strategy in the promotion of phytosterol biotransformation.

Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are generally produced by the biotransformation of phytosterols in Mycobacterium. The AD (D) production increases when the strain has high NAD+/NADH ratio. To enhance the AD (D) production in Mycobacterium neoaurum TCCC 11978 (MNR M3), a rational strategy was developed through overexpression of a gene involved in the phytosterol degradation pathway; NAD+ was generated as well. Proteomic analysis of MNR cultured with and without phytosterols showed that the steroid C27-monooxygenase (Cyp125-3), which performs sequential oxidations of the sterol side chain at the C27 position and has the oxidative cofactor of NAD+ generated, played an important role in the phytosterol biotransformation process of MNR M3. To improve the productivity of AD (D), the cyp125-3 gene was overexpressed in MNR M3. The specific activity of Cyp125-3 in the recombinant strain MNR M3C3 was improved by 22% than that in MNR M3. The NAD+/NADH ratio in MNR M3C3 was 131% higher than that in the parent strain. During phytosterol biotransformation, the conversion of sterols increased from 84 to 96%, and the yield of AD (D) by MNR M3C3 was increased by approximately 18% for 96h fermentation. This rational strain modification strategy may also be applied to develop strains with important application values for efficient production of cofactor-dependent metabolites.

Identification of 7α,24-dihydroxy-3-oxocholest-4-en-26-oic and 7α,25-dihydroxy-3-oxocholest-4-en-26-oic acids in human cerebrospinal fluid and plasma

Dihydroxyoxocholestenoic acids are intermediates in bile acid biosynthesis. Here, using liquid chromatography – mass spectrometry, we confirm the identification of 7α,24-dihydroxy-3-oxocholest-4-en-26-oic and 7α,25-dihydroxy-3-oxocholest-4-en-26-oic acids in cerebrospinal fluid (CSF) based on comparisons to authentic standards and of 7α,12α-dihydroxy-3-oxocholest-4-en-26-oic and 7α,x-dihydroxy-3-oxocholest-4-en-26-oic (where hydroxylation is likely on C-22 or C-23) based on exact mass measurement and multistage fragmentation. Surprisingly, patients suffering from the inborn error of metabolism cerebrotendinous xanthomatosis, where the enzyme CYP27A1, which normally introduces the (25 R)26-carboxylic acid group to the sterol side-chain, is defective still synthesise 7α,24-dihydroxy-3-oxocholest-4-en-26-oic acid and also both 25 R- and 25 S-epimers of 7α,12α-dihydroxy-3-oxocholest-4-en-26-oic acid. We speculate that the enzymes CYP46A1 and CYP3A4 may have C-26 carboxylase activity to generate these acids. In patients suffering from hereditary spastic paraplegia type 5 the CSF concentrations of the 7α,24- and 7α,25-dihydroxy acids are reduced, suggesting an involvement of CYP7B1 in their biosynthesis in brain.

Microbial catabolism of sterols: focus on the enzymes that transform the sterol 3β-hydroxy-5-en into 3-keto-4-en.

An overview on the microbial sterol catabolism is described with a focus on the catabolic step of the 3β-hydroxy-5-en structure. Cholesterol oxidase transforms this structure into the corresponding 3-keto-4-en feature, and thus initiates the sterol molecule catabolism. The oxidase has been found in a large number of microorganisms, especially in Actinobacteria as species of Rhodococcus and Streptomyces. Other Actinobacteria as species of Mycobacterium and Nocardia possess NAD(P)-dependent dehydrogenase for this catabolic step. In Rhodococcus jostii, oxidation of the C26 of the sterol side chain is the initiating step. The resulting stenone or sterol-C26-oic acid is then catabolized according to two subpathways: cleavage of the sterol side chain and degradation of the steroid nucleus. Divergent items concerned with the enzymes that transform the sterol 3β-hydroxy-5-en are discussed.

The Role of fadD19 and echA19 in Sterol Side Chain Degradation by Mycobacterium smegmatis.

Mycobacteria are able to degrade natural sterols and use them as a source of carbon and energy. Several genes which play an important role in cholesterol ring degradation have been described in Mycobacterium smegmatis. However, there are limited data describing the molecular mechanism of the aliphatic side chain degradation by Mycobacterium spp. In this paper, we analyzed the role of the echA19 and fadD19 genes in the degradation process of the side chain of cholesterol and β-sitosterol. We demonstrated that the M. smegmatis fadD19 and echA19 genes are not essential for viability. FadD19 is required in the initial step of the biodegradation of C-24 branched sterol side chains in Mycobacterium smegmatis mc2155, but not those carrying a straight chain like cholesterol. Additionally, we have shown that echA19 is not essential in the degradation of either substrate. This is the first report, to our knowledge, on the molecular characterization of the genes playing an essential role in C-24 branched side chain sterol degradation in M. smegmatis mc2155.

Cholesterol Analogs with Degradation-resistant Alkyl Side Chains Are Effective Mycobacterium tuberculosis Growth Inhibitors

Cholest-4-en-3-one, whether added exogenously or generated intracellularly from cholesterol, inhibits the growth ofMycobacterium tuberculosiswhen CYP125A1 and CYP142A1, the cytochrome P450 enzymes that initiate degradation of the sterol side chain, are disabled. Here we demonstrate that a 16-hydroxy derivative of cholesterol, which was previously reported to inhibit growth ofM. tuberculosis, acts by preventing the oxidation of the sterol side chain even in the presence of the relevant cytochrome P450 enzymes. The finding that (25R)-cholest-5-en-3β,16β,26-triol (1) (and its 3-keto metabolite) inhibit growth suggests that cholesterol analogs with non-degradable side chains represent a novel class of anti-mycobacterial agents. In accord with this, two cholesterol analogs with truncated, fluorinated side chains have been synthesized and shown to similarly block the growth in culture ofM. tuberculosis.

GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway.

Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules produced by solanaceous species. They contribute to pathogen defence but are toxic to humans and considered as anti-nutritional compounds. Here we show that GLYCOALKALOID METABOLISM 9 (GAME9), an APETALA2/Ethylene Response Factor, related to regulators of alkaloid production in tobacco and Catharanthus roseus, controls SGA biosynthesis. GAME9 knockdown and overexpression in tomato and potato alters expression of SGAs and upstream mevalonate pathway genes including the cholesterol biosynthesis gene STEROL SIDE CHAIN REDUCTASE 2 (SSR2). Levels of SGAs, C24-alkylsterols and the upstream mevalonate and cholesterol pathways intermediates are modified in these plants. Δ(7)-STEROL-C5(6)-DESATURASE (C5-SD) in the hitherto unresolved cholesterol pathway is a direct target of GAME9. Transactivation and promoter-binding assays show that GAME9 exerts its activity either directly or cooperatively with the SlMYC2 transcription factor as in the case of the C5-SD gene promoter. Our findings provide insight into the regulation of SGA biosynthesis and means for manipulating these metabolites in crops.

Cholesterol's Aliphatic Side Chain Structure Modulates Membrane Properties

The influence of the length of the cholesterol alkyl chain on essential membrane properties such as lipid ordering (condensation), lateral diffusion, membrane permeability, and membrane domain formation was studied using a series of synthetic cholesterol derivatives without a side chain or with an iso-branched chain bearing 5 to 14 carbons in length. We found that cholesterol's aliphatic side chain is crucial for all of the membrane properties investigated. First, we detected that the side chain is responsible for more than half of the phospholipid condensation in bilayers. Second, the length of the sterol side chain strongly determines membrane permeation, since a slightly longer or shorter side chain renders the bilayer significantly more permeable. Third, lateral lipid and sterol diffusion highly depends on the side chain length. Fourth, the length of the cholesterol side chain also determines the lateral organization of lipids in raft mixtures. While all tested sterols induce domain formation, the lipid distribution between the liquid disordered or raft phase is strongly dependent on side chain length. Fifth, the sterol side chain also influences the partitioning of a transmembrane peptide domain into liquid-ordered or liquid-disordered domains. Therefore, in addition to the flat tetracyclic ring system of cholesterol, the iso-branched side chain of cholesterol plays a key role in producing the relevant membrane properties of eukaryotic cells that result in an optimal interaction with phospholipids and presumably with membrane proteins. Sterols bearing an unbranched alkyl side chain of varying length also influence important properties of lipid membranes. However, the extent of this impact is lower compared with that measured for the respective branched iso-side chain sterols. Obviously, sterols having a branched iso-chains with two terminal methyl groups exhibit altered cholesterol-phospholipid-interactions compared to molecules with a straight unbranched chain.

Arylpiperidines as a new class of oxidosqualene cyclase inhibitors

The cyclization of oxidosqualene to lanosterol, catalyzed by the enzyme oxidosqualene cyclase (OSC), goes through a number of carbocationic high energy intermediates (HEI), and mimicking these intermediates is a promising approach for the development of OSC inhibitors. 3-Arylpiperidines (or tetrahydropyridines) were designed as steroidomimetic rings A + C equivalents containing two protonable amino groups for mimicking both the pro-C4 HEI and the pro-C20 HEI of the OSC-mediated cyclization cascade. Inhibitory activity is strongly dependent on the nature of the lipophilic substituent representing an equivalent of the sterol side chain. Here aromatic residues (substituted benzyl, cinnamyl, naphthylmethyl) were found to be most suitable. Docking experiments on a first optimized 3-arylpiperidine compound led to an isomeric 4-arylpiperidine with submicromolar activity on human OSC. This inhibitor reduced total cholesterol biosynthesis in a cellular assay with an IC50 value of 0.26 μM.

The influence of cholesterol precursor – desmosterol – on artificial lipid membranes

The disorders in cholesterol biosynthesis pathway and various diseases manifest in the accumulation of cholesterol precursors in the human tissues and cellular membranes. In this paper the effect of desmosterol – one of cholesterol precursors – on model lipid membranes was studied. The investigations were performed for binary SM/desmo and POPC/desmo and ternary SM/POPC/desmo monolayers. Moreover, the experiments based on the gradual substitution of cholesterol by desmosterol in SM/POPC/chol=1:1:1 system were done. The obtained results allowed one to conclude that desmosterol is of lower domains promoting and stabilizing properties and packs less tightly with the lipids in monolayers. Moreover, desmosterol probably could replace cholesterol in model membranes, but only at its low proportion in the system (2%), however, at a higher degree of cholesterol substitution a significant decrease of the monolayer stability and packing and alterations in the film morphology were detected. The results collected in this work together with those from previous experiments allowed one to analyze the effect of a double bond in the sterol side chain as well as its position in the ring system on membrane activity of the molecule and to verify Bloch hypothesis.

Influence of the sterol aliphatic side chain on membrane properties: a molecular dynamics study.

Following a recent experimental investigation of the effect of the length of the alkyl side chain in a series of cholesterol analogues (Angew. Chem., Int. Ed., 2013, 52, 12848-12851), we report here an atomistic molecular dynamics characterization of the behaviour of methyl-branched side chain sterols (iso series) in POPC bilayers. The studied sterols included androstenol (i-C0-sterol) and cholesterol (i-C8-sterol), as well as four other derivatives (i-C5, i-C10, i-C12 and i-C14-sterol). For each sterol, both subtle local effects and more substantial differential alterations of membrane properties along the iso series were investigated. The location and orientation of the tetracyclic ring system is almost identical in all compounds. Among all the studied sterols, cholesterol is the sterol that presents the best matching with the hydrophobic length of POPC acyl chains, whereas longer-chained sterols interdigitate into the opposing membrane leaflet. In accordance with the experimental observations, a maximal ordering effect is observed for intermediate sterol chain length (i-C5, cholesterol, i-C10). Only for these sterols a preferential interaction with the saturated sn-1 chain of POPC (compared to the unsaturated sn-2 chain) was observed, but not for either shorter or longer-chained derivatives. This work highlights the importance of the sterol alkyl chain in the modulation of membrane properties and lateral organization in biological membranes.

Sterols of Saccharomyces cerevisiae erg6 Knockout Mutant Expressing the Pneumocystis carinii S-Adenosylmethionine:Sterol C-24 Methyltransferase.

The AIDS-associated lung pathogen Pneumocystis is classified as a fungus although Pneumocystis has several distinct features such as the absence of ergosterol, the major sterol of most fungi. The Pneumocystis carinii S-adenosylmethionine:sterol C24-methyltransferase (SAM:SMT) enzyme, coded by the erg6 gene, transfers either one or two methyl groups to the C-24 position of the sterol side chain producing both C28 and C29 24-alkylsterols in approximately the same proportions, whereas most fungal SAM:SMT transfer only one methyl group to the side chain. The sterol compositions of wild-type Sacchromyces cerevisiae, the erg6 knockout mutant (Δerg6), and Δerg6 expressing the P. carinii or the S. cerevisiae erg6 gene were analyzed by a variety of chromatographic and spectroscopic procedures to examine functional complementation in the yeast expression system. Detailed sterol analyses were obtained using high performance liquid chromatography and proton nuclear magnetic resonance spectroscopy ((1)H-NMR). The P. carinii SAM:SMT in the Δerg6 restored its ability to produce the C28 sterol ergosterol as the major sterol, and also resulted in low levels of C29 sterols. This indicates that while the P. carinii SAM:SMT in the yeast Δerg6 cells was able to transfer a second methyl group to the side chain, the action of Δ(24(28)) -sterol reductase (coded by the erg4 gene) in the yeast cells prevented the formation and accumulation of as many C29 sterols as that found in P. carinii.

Sterol side chain reductase 2 is a key enzyme in the biosynthesis of cholesterol, the common precursor of toxic steroidal glycoalkaloids in potato.

Potatoes (Solanum tuberosum) contain α-solanine and α-chaconine, two well-known toxic steroidal glycoalkaloids (SGAs). Sprouts and green tubers accumulate especially high levels of SGAs. Although SGAs were proposed to be biosynthesized from cholesterol, the biosynthetic pathway for plant cholesterol is poorly understood. Here, we identify sterol side chain reductase 2 (SSR2) from potato as a key enzyme in the biosynthesis of cholesterol and related SGAs. Using in vitro enzyme activity assays, we determined that potato SSR2 (St SSR2) reduces desmosterol and cycloartenol to cholesterol and cycloartanol, respectively. These reduction steps are branch points in the biosynthetic pathways between C-24 alkylsterols and cholesterol in potato. Similar enzymatic results were also obtained from tomato SSR2. St SSR2-silenced potatoes or St SSR2-disrupted potato generated by targeted genome editing had significantly lower levels of cholesterol and SGAs without affecting plant growth. Our results suggest that St SSR2 is a promising target gene for breeding potatoes with low SGA levels.

Identification of gene expression profiles in the actinomycete Gordonia neofelifaecis grown with different steroids.

Gordonia neofelifaecis NRRL B-59395 was initially isolated from the fresh feces of a clouded leopard based on its ability to degrade cholesterol. The transcriptome profiles of G. neofelifaecis NRRL B-59395 grown with cholesterol, androstenedione (AD), and pyruvic acid were compared by RNA-Seq. The sterol catabolic genes are highly conserved in G. neofelifaecis, Rhodococcus jostii RHA1, and Mycobacterium tuberculosis. The RNA-Seq results indicated that the genes involved in the sterol side chain cleavage were exclusively induced by cholesterol, while the genes involved in the degradation of rings A/B and C/D were up-regulated by both cholesterol and AD. It appears that the induction mechanisms for the genes responsible for side chain cleavage and those for degradation of rings are different. There are approximately 21 genes encoding transporter proteins that are differentially expressed in cholesterol or AD compared with pyruvic acid. The genes camABCD and camM encode two systems that take up cholate, and they have been shown to be cholesterol- and AD-inducible. The potential biological functions of other differentially expressed genes are also discussed. These results will promote the functional characterization of the sterol catabolic genes and also provide important clues in understanding the mechanisms of their gene expression, and they may help us understand the mechanism underlying microbial cholesterol catabolism.

Effect of sterol side chain on ion channel formation by amphotericin B in lipid bilayers.

Amphotericin B (AmB) is one of the most efficient antimycotic drugs used in clinical practice. AmB interacts with membrane sterols increasing permeability of fungal membranes; however, it is still unclear how AmB selectively recognizes the fungal sterol, ergosterol (Erg), over other sterols in cell membranes. In this study, we investigated the effect of an Erg side chain on AmB activity by testing a series of Erg analogues that shared the same alicyclic structure as Erg but varied in the side chain structure by using the K(+) influx assay. The results clearly showed that the sterol side chain is essential for AmB selectivity toward Erg and for the activity of AmB-sterol ion channels. In agreement with our previous findings showing the direct interaction between the drug and Erg, these data suggested that AmB directly recognizes the sterol side chain structure, consequently promoting the formation of ion channels by AmB. Furthermore, the C24 methyl group and Δ22 double bond in the side chain of Erg are equally important for the interaction with AmB. Conformational analysis revealed that the C24 methyl group contributes to the interaction by increasing the van der Waals (VDW) contact area of the side chain, while the Δ22 double bond restricts the side chain conformation to maximize the VDW contact with the rigid AmB aglycone. This study provides direct experimental evidence of the mechanism of AmB selectivity toward fungal Erg.

Triple Shifts and Thioether Assistance in Rearrangements Associated with an Unusual Biomethylation of the Sterol Side Chain

Quantum chemical calculations (B3LYP and MP2) are described for the formation and rearrangements of carbocations derived from the biological methylation reaction that produces 24-propyl sterols in pelagophyte algae. Previous mechanistic proposals are discussed in light of the results of these calculations. Of particular note is the prediction of a new triple-shift rearrangement that is inherently preferred for the biosynthetically relevant carbocations. Our calculations also reveal how these reactions may be affected by intermolecular interactions with S-adenosylmethionine.

Molecular characterization of ltp3 and ltp4, essential for C24-branched chain sterol-side-chain degradation in Rhodococcus rhodochrous DSM 43269

A previously identified sterol catabolic gene cluster is widely dispersed among actinobacteria, enabling them to degrade and grow on naturally occurring sterols. We investigated the physiological roles of various genes by targeted inactivation in mutant RG32 of Rhodococcus rhodochrous, which selectively degrades sterol side-chains. The ltp3 and ltp4 deletion mutants were each completely blocked in side-chain degradation of β-sitosterol and campesterol, but not of cholesterol. These results indicated a role for ltp3 and ltp4 in the removal of C24 branches specifically. Bioinformatic analysis of the encoded Ltp3 and Ltp4 proteins revealed relatively high similarity to thiolase enzymes, typically involved in β-oxidation, but the catalytic residues characteristic for thiolase enzymes are not conserved in their amino acid sequences. Removal of the C24-branched side-chain carbons of β-sitosterol was previously shown to proceed via aldolytic cleavage rather than by β-oxidation. Our results therefore suggest that ltp3 and ltp4 probably encode aldol-lyases rather than thiolases. This is the first report, to our knowledge, on the molecular characterization of genes with specific and essential roles in carbon-carbon bond cleavage of C24-branched chain sterols in Rhodococcus strains, most likely acting as aldol-lyases. The results are a clear contribution to our understanding of sterol degradation in actinobacteria.

Influence of hydroxypropyl-β-cyclodextrin on phytosterol biotransformation by different strains of Mycobacterium neoaurum

Cyclodextrins (CDs) can improve productivity in the biotransformation of steroids by increasing conversion rate, conversion ratio, or substrate concentration. However, little is known of the proportion of products formed by multi-catabolic enzymes, e.g., via sterol side chain cleavage. Using three strains with different androst-1,4-diene-3,17-dione (ADD) to androst-4-ene-3,17-dione (AD) ratios, Mycobacterium neoaurum TCCC 11028 (MNR), M. neoaurum TCCC 11028 M1 (MNR M1), and M. neoaurum TCCC 11028 M3 (MNR M3), we found that hydroxypropyl-β-cyclodextrin (HP-β-CD) can appreciably increase the ratio of ADD to AD, the reaction rate, and the molar conversion. In the presence of HP-β-CD, conversion of 0.5 g/L of phytosterol (PS) was 2.4, 2.4, and 2.3 times higher in the MNR, MNR M1, and MNR M3 systems, respectively, than in the controls. The ADD proportion increased by 38.4, 61.5, and 5.9 % compared with the control experiment, which resulted in a strong shift in the ADD/AD ratio in the ADD direction. Our results imply that the three PS-biotransforming strains cause efficient side chain degradation of PS, and the increased conversion of PS when using HP-β-CD may be associated with the higher PS concentration in each case. A similar solubilizing effect may not induce a prominent influence on the ADD/AD ratio. However, the different activities of the Δ¹-dehydrogenase of PS-biotransforming strains result in different incremental percentage yields of ADD and ADD/AD ratio in the presence of HP-β-CD.

Evolutionarily conserved Delta(25(27))-olefin ergosterol biosynthesis pathway in the alga Chlamydomonas reinhardtii.

Ergosterol is the predominant sterol of fungi and green algae. Although the biosynthetic pathway for sterol synthesis in fungi is well established and is known to use C24-methylation-C24 (28)-reduction (Δ(24(28))-olefin pathway) steps, little is known about the sterol pathway in green algae. Previous work has raised the possibility that these algae might use a novel pathway because the green alga Chlamydomonas reinhardtii was shown to possess a mevalonate-independent methylerythritol 4-phosphate not present in fungi. Here, we report that C. reinhardtii synthesizes the protosterol cycloartenol and converts it to ergosterol (C24β-methyl) and 7-dehydroporiferasterol (C24β-ethyl) through a highly conserved sterol C24- methylation-C25-reduction (Δ(25(27))-olefin) pathway that is distinct from the well-described acetate-mevalonate pathway to fungal lanosterol and its conversion to ergosterol by the Δ(24(28))-olefin pathway. We isolated and characterized 23 sterols by a combination of GC-MS and proton nuclear magnetic resonance spectroscopy analysis from a set of mutant, wild-type, and 25-thialanosterol-treated cells. The structure and stereochemistry of the final C24-alkyl sterol side chains possessed different combinations of 24β-methyl/ethyl groups and Δ(22(23))E and Δ(25(27))-double bond constructions. When incubated with [methyl-(2)H(3)]methionine, cells incorporated three (into ergosterol) or five (into 7-dehydroporiferasterol) deuterium atoms into the newly biosynthesized 24β-alkyl sterols, consistent only with a Δ(25(27))-olefin pathway. Thus, our findings demonstrate that two separate isoprenoid-24-alkyl sterol pathways evolved in fungi and green algae, both of which converge to yield a common membrane insert ergosterol.

Proximal Ligand Electron Donation and Reactivity of the Cytochrome P450 Ferric–Peroxo Anion

CYP125 from Mycobacterium tuberculosis catalyzes sequential oxidation of the cholesterol side-chain terminal methyl group to the alcohol, aldehyde, and finally acid. Here, we demonstrate that CYP125 simultaneously catalyzes the formation of five other products, all of which result from deformylation of the sterol side chain. The aldehyde intermediate is shown to be the precursor of both the conventional acid metabolite and the five deformylation products. The acid arises by protonation of the ferric-peroxo anion species and formation of the ferryl-oxene species, also known as Compound I, followed by hydrogen abstraction and oxygen transfer. The deformylation products arise by addition of the same ferric-peroxo anion to the aldehyde intermediate to give a peroxyhemiacetal that leads to C-C bond cleavage. This bifurcation of the catalytic sequence has allowed us to examine the effect of electron donation by the proximal ligand on the properties of the ferric-peroxo anion. Replacement of the cysteine thiolate iron ligand by a selenocysteine results in UV-vis, EPR, and resonance Raman spectral changes indicative of an increased electron donation from the proximal selenolate ligand to the iron. Analysis of the product distribution in the reaction of the selenocysteine substituted enzyme reveals a gain in the formation of the acid (Compound I pathway) at the expense of deformylation products. These observations are consistent with an increase in the pK(a) of the ferric-peroxo anion, which favors its protonation and, therefore, Compound I formation.