fosfomycin , or ( 1r,2s)-1,2-epoxypropyl - phosphonic acid , is an antibiotic used in the treatment of lower urinary tract infections(1 ) and is effective against methicillin - resistant(2 ) and vancomycin - resistant(3 ) strains of staphylococcus aureus . it targets the first committed step in cell wall biosynthesis by inhibiting the enzyme udp - glcnac-3-o - enolpyruvyltransferase ( mura ) . while epoxide rings are found in many natural products , the way in which the fosfomycin epoxide ring is biosynthesized by microbes such as streptomyces wedmorensis and pseudomonas syringae may be unique . unlike other characterized epoxidases that form epoxides through oxygen atom insertion , hydroxypropylphosphonic acid epoxidase ( hppe ) forms fosfomycin by catalyzing an oxidative cyclization reaction with retention of the substrate hydroxyl oxygen atom ( scheme 1a ) . despite one report(11 ) that hppe has some catalytic activity with zinc , recent studies show that under defined conditions , zn  hppe does not produce fosfomycin , reaffirming this enzyme as a member of the mononuclear nonheme iron superfamily.(12 ) hppe shares several features with other members of this superfamily , including the use of the so - called 2-his-1-carboxylate facial triad [ his2(glu / asp ) ] to coordinate an iron atom within a conserved -barrel fold also known as a cupin fold . with highest structural similarity to the mononuclear iron enzyme hydroxyethylphosphonate dioxygenase,(16 ) hppe is an unusual tetramer with four -barrels intertwined with four -helical domains , creating four active sites at each of the  interfaces ( figure 1).(15 ) the substrate ( s)-2-hydroxypropylphosphonic acid ( s - hpp ) binds directly to the iron center in these active sites , triggering conformational changes in the -hairpin , or cantilever hairpin , which is directly above each facial triad.(15 ) while other mononuclear iron enzymes can undergo conformational changes when substrates bind , the use of these -strands is unique to hppe . this conformational change buries the hydrophobic portion of the substrate , reducing solvent accessibility of the active site and thus protecting high - energy iron  oxygen intermediates , such as a superoxide intermediate ( fe  o2),(20 ) which may form during catalysis . for complete turnover , hppe also requires two exogenous electrons , and unlike many members of this superfamily , -ketoglutarate does not serve this function . instead , these electrons are derived from nad(p)h and shuttled to the active site by a reductase in vivo . another fascinating feature of hppe is that it recognizes both s - hpp and its enantiomeric substrate r - hpp and is an efficient catalyst with respect to both reactions . rather , this stereoisomer exclusively yields a ketone product , 2-oxopropylphosphonic acid ( scheme 1b ) . to probe the mechanism of regiospecificity of hppe catalysis , both enantiomers of the substrate were synthesized incorporating fluorines in place of the c1 methylene hydrogens ( s - fhpp and r - fhpp).(22)s - fhpp is a strong competitive inhibitor of hppe activity , while r - fhpp is a substrate and is converted to 1-difluoro-2-oxopropylphosphonic acid ( scheme 1c , d).(22 ) this result suggests that the reaction path is determined by the regiospecificity of hydrogen atom abstraction ; a c1 hydrogen atom is abstracted from the s - hpp substrate , and a c2 hydrogen atom is abstracted from r - hpp . a radical centered at c1 would then yield the epoxide product , whereas the c2 radical yields the ketone.(22 ) the hppe tetramer is shown colored by molecule in blue , magenta , yellow , and green , with strand 1 and the cantilever hairpin colored separately in cyan , pink , wheat , and limon , respectively . iron atoms are shown as spheres ( rust ) , and iron ligands are in sticks : carbon ( gray ) , nitrogen ( blue ) , oxygen ( red ) . although the results from these fluorinated analogues provide an explanation for why a different product is formed from each stereoisomer , we sought to understand why the enzyme would abstract a different hydrogen atom in each case . first , it is important to identify where dioxygen binds to the iron in the enzyme active site . while it has been proposed that dioxygen binds iron at the only open coordination site that exists when s - hpp is bound in the bidentate conformation,(15 ) here we use no as a dioxygen mimic to obtain experimental evidence for this binding site . the next question is whether the r- and s - stereoisomers bind the iron active site in a different fashion , and , if so , what is the cause of differential binding ? if they bind in the same fashion , can differences in c  h bond dissociation energies ( calculated to be 96.5 and 89.0 kcal mol for s - hpp ( c1h ) and r - hpp ( c2h ) , respectively(6 ) ) explain the observed regiospecificity ? to address these questions , we have solved a structure at 2.1  resolution of co(ii)hppe with the r - stereoisomer of substrate bound . additionally , we have determined the structure of the substrate enantiomer in complex with fe(ii)hppe at 3.0  resolution . these structures reveal that r - hpp has a binding mode similar to that of s - hpp but differs with respect to the relative orientation of the c1 , c2 , and c3 positions . furthermore , the structures illustrate that hydrogen atom accessibility to the proposed iron - superoxo intermediate species is sufficient to explain the regiospecificity of the hppe - catalyzed reactions . cobalt(ii ) chloride was purchased from hampton research ( aliso viejo , ca ) , diethylamine nonoate was from cayman chemical ( ann arbor , mi ) , and other reagents were from sigma - aldrich ( st . wild - type and selenomethione - derivatized apo - hppe was expressed and purified as previously described . apo - protein was concentrated to 30 mg / ml and dialyzed into 0.02 m tris - hcl , at ph 8.0 . enzyme activity of apo - hppe reconstituted with fe(ii)(nh4)2(so4)26h2o was determined using a p nmr assay . r - hpp ( 0.6 l , 0.2 m ) was added to fe(ii)hppe crystals placed over a reservoir of soaking buffer solution ( 0.1 m hepes , ph 7.5 , 2.5 m ammonium sulfate ) and allowed to diffuse into the crystals for 14 h. the crystals were flash - cooled in liquid nitrogen following a 30 s transfer to a cryo - protectant buffer solution ( 0.1 m tris - hcl , ph 8.0 , 2.0 m ammonium sulfate , 30% ( w / v ) xylitol ) . crystals of r - hpp in complex with co(ii)hppe were obtained by soaking this substrate into co(ii)hppe crystals.(15 ) the hexagonal crystals used for soaking were grown by mixing 2.0 l of 30 mg / ml selenomethionine  apo - hppe solution in 0.02 m tris  hcl ph 8.0 with 2.0 l of precipitant buffer solution ( 0.1 m tris  hcl , ph 8.5 , 2.0 m ammonium sulfate ) using the hanging - drop vapor diffusion method at room temperature . cocl2 ( 0.3 l , 0.1 m ) solution was added to the drop , and the so - called  tris  co(ii)hppe  crystals grew in 24 h. r - hpp  co(ii)hppe crystals were obtained from tris co(ii)hppe crystals by first transferring crystals to soaking buffer solution to displace any tris molecules coordinating co(ii ) . the removal of tris was verified through a structure determination ( data not shown ) . the co(ii)hppe crystals were transferred to 4.0 l of fresh soaking buffer solution for 10 min . r - hpp ( 1.0 l , 2.5 m ) was added to the drop , and the resulting solution was placed over a reservoir of soaking solution for additional 14 h. the crystals were transferred to cryo - protectant buffer solution for 30 s and cooled in liquid nitrogen . the ternary complex ( s - hpp s - hpp ( 0.6 l , 0.2 m ) was added to fe(ii)hppe crystals placed over a reservoir of soaking buffer solution . nitric oxide ( no ) was introduced to the well with s - hpp  fe(ii)hppe crystals by liberation from diethylamine nonoate . after 1 h of equilibration , no gas ( 2.0 ml ) was injected into the well for additional 2 h. crystals were then transferred to cryo - protectant buffer solution and flash - cooled in liquid nitrogen . fe(ii)hppe  no were collected at the stanford synchrotron radiation lightsource and the advanced photon source , respectively . the r - hpp  co(ii)hppe data set was collected at the advanced light source . the data were integrated and scaled in denzo and scalepack,(25 ) respectively ( supporting information table 1 ) . crystals of s - hpp fe(ii)hppe  no ( p42212 ; a , b = 111.66  , c = 152.16  ) and r - hpp  fe(ii)hppe ( p42212 ; a , b = 111.67  , c = 152.81  ) , with three molecules in the asymmetric unit , were isomorphous with our previous crystals of s - hpp  fe(ii)hppe ( p42212 ; a , b = 111.65  , c = 152.07  ) . thus , these structures were solved by rigid body refinement in cns(26 ) using protein atoms from s - hpp model building was done in coot,(27 ) and iterative refinement steps were accomplished using cns . topology and parameter files for r - hpp were obtained using hic - up.(28 ) parameter and topology files for the iron - no coordination sphere were developed using the bond distances and angles from high - resolution x - ray structures of nonheme iron model complexes with no bound to iron.(29 ) to allow movement of atoms away from the average values , weak restraints were used . the cns protocol included simulated annealing against a maximum likelihood function target , positional refinement , and b - factor refinement ; noncrystallographic symmetry restraints and sigma cutoff were not used . because the r - hpp  co(ii)hppe crystal , consisting of two molecules in the asymmetric unit ( p6522 ; a , b = 86.30  , c = 219.1  ) was isomorphous with our previous crystals of tris  co(ii)hppe ( p6522 ; a , b = 86.36  , c = 220.17  ) , the r - hpp  co(ii)hppe structure was solved by rigid body refinement in cns using only protein atoms from the refined tris  co(ii)hppe structure(15 ) as an initial model ( pdb code 1zzc ) . iterative model refinement was performed in cns using the protocol described above . manual adjustment of the model was performed using xfit.(30 ) structures of r - hpp fe(ii)hppe  no were analyzed using 2fo  fc composite omit maps , and ramachandran geometries were analyzed with procheck . the results from procheck indicate that the r - hpp  co(ii)hppe structure has 89.4% of the residues in the most favored and 10.6% of the residues in the additionally allowed regions of the ramachandran plot . for r - hpp  fe(ii)hppe structure , the ramachandran plot indicates that 87.7% of the residues are in the most favored and 12.3% of the residues are in the additionally allowed regions . the ramachandran plot for s - hpp fe(ii)hppe  no structure indicates that 86.2% of the residues are in the most favored , 13.6% of the residues are in the additionally allowed regions , and 0.2% of the residues in the generously allowed regions . chain a of the r - hpp  co(ii)hppe structure contains residues 5198 , while other chains consist of residues 6198 . the crystal structure of co(ii)hppe in complex with r - hpp was solved to 2.1  resolution ( figure 2a , supporting information table 1 ) . co(ii)hppe is catalytically inert under aerobic conditions(9 ) and allows us to observe at high resolution an enzyme state that mimics the form of the enzyme prior to dioxygen binding . we have also solved this same structure using the native fe(ii ) metal ion in the absence of dioxygen ( figure 2b ) , but to lower resolution ( 3.0  ) . we find that the overall structures are similar , with root - mean - square deviations of 0.270.71  . in each case , substrate can be unambiguously positioned in the active site due to the presence of 810  electron density peaks that identify the position of the hpp phosphorus atom ( figure 2 ) . as was the case for the s - enantiomer , r - hpp binds to the active site metal in a bidentate binding mode coordinated by both the c2 hydroxyl and the c1 phosphonate moieties ( figure 2a  d ; also see supporting information figure 1a1e).(15 ) the bidentate binding mode is observed in both the fe(ii ) and the co(ii ) structures ( see supporting information table 2 for a complete list of structures ) . structures of hppe with r - hpp or s - hpp bound in the active site . ( c ) bidentate binding mode for s - hpp in the co(ii)hppe structure ( 1zzb monomer b ) . ( d ) bidentate binding mode for s - hpp in the fe(ii)hppe structure ( 1zz8 monomer c ) . substrate and protein residues are shown as sticks ( carbon ( gray ) , oxygen ( red ) , nitrogen ( blue ) , phosphorus ( purple ) ) , water molecules as spheres ( cyan ) , co(ii ) as a sphere ( magenta ) , and fe(ii ) as a sphere ( rust ) . the 2fo  fc maps ( blue mesh ) are contoured at 1.0  , and the fo  fc omit - maps ( green mesh ) of the substrate indicating the position of the phosphorus are contoured at 8.0 , 10.0 , 9.0 , and 8.0  , respectively . hppe appears to use an induced - fit mechanism to protect high - energy iron oxygen species formed during catalysis.(15 ) a -hairpin called the cantilever hairpin ( residues 90107 ) responds to the physiological substrate s - hpp when bound to the enzyme in the correct orientation by moving in and sealing off the top portion of the active site , leaving only a small opening for dioxygen to enter.(15 ) comparison of s - hpp fe(ii)hppe structures with the alternative r - hpp substrate shows a similar response ; the cantilever hairpin closes down and becomes more ordered upon bidentate substrate binding . in fact , in the closed conformation , the cantilever hairpin in the r - hpp  fe(ii)hppe structure superimposes well with that in the s - hpp  fe(ii)hppe structure ( pdb i d 1zz8 ) ( figure 3 ) , producing very similar interactions between the protein and s- and r - hpp substrates . a number of hydrogen bonds and electrostatic interactions made by the protein with the substrate are contributed from residues of the cantilever hairpin ( arg97 and tyr105 ) . other contacts are made from other -barrel residues as well as one residue of the helical domain ( lys23 ) . in the bidentate conformation of both s- and r - hpp , all hydrogen - bonding interactions are made to the phosphonate moiety . in every structure , the phosphonate substituent charge interactions with lys23 from the helical domain and hydrogen - bonding interactions with tyr105 and asn135 of the -barrel ( supporting information figure 1 ) . in the majority of both the s - hpp and the r - hpp bound fe - hppe structures , arg97 is ordered and contacts the phosphonate moiety both directly and indirectly via a water molecule to further stabilize the additional charge in the active site ( supporting information figure 1c comparison of the cantilever hairpins ( -strands labeled 1 and 2 ) of the r - hpp  fe(ii)hppe structure ( green ) and the s - hpp  fe(ii)hppe structure ( cyan ) . bound substrate and selected residues are shown as sticks , and iron atoms are shown as spheres ( rust ) . with the hairpin closed , there is space available for oxygen to enter the active site in a cavity created at the interface of the helical and -barrel domains.(15 ) this cavity leads to the only open coordination site in any of the substrate - bound structures , a position trans to his180 . to obtain experimental evidence that this site is in fact the oxygen binding site , fe(ii)hppe  no at 2.85  resolution reveals bidentate coordination of the substrate to the fe(ii ) center via the c2 hydroxyl and the phosphonate oxygen in all three molecules in the asymmetric unit . in one of the three monomers , there is density only for a water molecule and not for no ( supporting information figure 1h ) . however , in the other two monomers ( monomers a , b ( fe  s - hpp  no ) ) , no is coordinated to the fe(ii ) center as seen by omit and positive difference electron density that is consistent with a diatomic molecule ( figure 4a , b ; also see supporting information figure 1f , g ) . when a water molecule is refined into the electron density of monomers a and b instead of no , positive difference electron density results , indicating the presence of a second atom ( figure 4b ) . when no is refined in this position , no positive or negative difference electron density appears , indicating that a diatomic molecule is the correct size . because the only difference between the s - hpp  fe(ii)no structure and the previous s - hpp bound fe hppe structures is the soaking of the crystals with no , we have modeled and refined no in this site in these two monomers . we find that , as predicted , no coordinates trans to the his180 iron ligand , presumably via the nitrogen atom in the site that was  open  in the substrate - bound structure . the fe n bond distances in the two no bound monomers are 1.64 and 1.66  , and the fe o bond angles are 160 and 161 , which are typical values for iron  nitrosyl interactions in model complexes.(29 ) the oxygen atom of the no is pointed toward c1 of the substrate at an average distance of 3.5  . c1 is the proposed carbon from which a hydrogen atom is abstracted.(9 ) while it was predicted previously that lys23 could interact with the active oxygen species,(15 ) here we find that the oxygen atom of no is far from the terminal amine of lys23 ( 4.8  in monomer a and 5.2  in the monomer b ) , although movement of this residue is possible . alternatively , lys23 may play its essential role(15 ) at some other stage in the mechanism . fe(ii)hppe is shown with the fe(ii ) atom ( rust ) as a sphere and substrate , residues , and no as sticks . an fo  fc omit map ( green mesh ) is contoured at 7.0 . ( b ) when water is refined in a position similar to that of the nitrogen of no in the active site , shown in figure 4a , positive fo fc difference density ( green mesh ) at 4.0  indicates that water is not large enough to account for the density . many of the hppe structures that have bound substrate also have highly ordered water molecules in the active site . of particular interest are water molecules , which are found in the r - hpp  hppe structures and lie near lys23 in a position trans to facial triad ligand his180 without coordinating the metal ( figure 2a , b ) . a water molecule was observed in a similar position in one of three active sites within the asymmetric unit of the previously determined s - hpp  fe(ii)hppe structure in tetragonal space group(15 ) ( figure 2d ) . there is a range of distances that waters can occupy in this general water binding region ( 2.53.9  ) . waters located at approximately 2.8  from the metal are in the same position as the terminal oxygen of no when it is bound in the open metal coordination site ( comparable to the second oxygen of dioxygen ) ( figure 2b , d ; also see supporting information figure 1e , i ) . waters at distances of 3.9  from the metal may represent a product water position ( figures 2a ; also see supporting information figure 1a , b ) . with experimental information from our no data supporting the assignment of the binding site for dioxygen on iron , we can examine the proposed mechanisms for regiospecificity by comparing our s - hpp and r - hpp bound structures . both enantiomers bind to the same general site on the metal in the active site of hppe . for s - hpp , its hydrophobic portion including the c2 methyl group is accommodated by a hydrophobic pocket on the enzyme , comprising residues phe182 , leu193 , and leu144 ( figure 5).(15 ) because the position of the c2 methyl moiety of the r - hpp molecule ( bidentate binding mode ) is within 1  of the corresponding position of the c2 methyl moiety of s - hpp ( figure 6a ) , the same hydrophobic binding pocket serves to accommodate the methyl moiety of the r - enantiomer ( figure 5 ) . even though the binding modes for both substrate enantiomers are similar , they are not identical ( figure 6a ) , and even small differences in orientations of c1 and c2 can have a significant impact in terms of accessibility of their hydrogens for abstraction by the reactive iron  oxygen intermediates . while hydrogen atoms are not discernible in electron density , the orientation of the sp carbon atoms ( i.e. , tetrahedral carbon atoms ) can be accurately modeled within the electron density . therefore , the positions of the hydrogen atoms covalently bound to these carbon atoms can be inferred . using the binding site of no to mark the likely location of the dioxygen binding site , the modeling of hydrogens suggests that for the s - hpp substrate , the pro - r hydrogen at c1 points toward this putative dioxygen binding site ( 3.5  between o of no and c1 ) , while the c2 hydrogen is oriented in the opposite direction ( 4.3  between o of no and c2 ) ( figure 6b ) . in contrast , for the r - hpp structures , the modeled c1 methylene hydrogens point away from ( 4.0  between o of no and c1 ) while the c2 hydrogen points toward the dioxygen binding site ( 3.1  between o of no and c2 ) ( figure 6c ) . thus , despite the similar bidentate binding modes , the orientation of the c1 and c2 carbons and their hydrogen atoms appear to be quite different . cantilever hairpin residues and residues interacting with the aliphatic portion of r - hpp ( l144 , l193 , and f182 ) are shown as sticks . iron is shown as a sphere ( rust ) . it is interesting to consider why r - hpp binds to the iron with such a difference in the positions of c1 and c2 as compared to s - hpp , and whether at this resolution we can be sure of this alternative conformation . when we compare the refined model of r - hpp ( green in figure 6c , d ) with a hypothetical conformation of r - hpp that is based on the s - substrate conformation ( wheat in figure 6d ) , we find that there is no electron density to support the placement of the methyl group on c2 in the conformation represented by the hypothetic model ( figure 6d ) . the alternative conformation is a much better fit to the density . in considering why the hypothetical conformation is not favored , modeling suggests that the methyl group on c2 would be close ( 2.8  ) to the phosphonic acid moiety in this conformation , producing a steric clash ( figure 6d ) . the electron density is consistent with an extended and staggered conformation of r - hpp in which the methyl moiety is farthest from the bulky phosphonic acid moiety ( figure 6c ; also see supporting information figure 2 ) . mononuclear iron enzymes catalyze a wide range of reactions from antibiotic biosynthesis to dna repair . while the products of the reactions vary , most mononuclear iron enzymes must accomplish the same tasks ( activate the iron center to facilitate dioxygen binding , protect high energy intermediates throughout the catalytic cycle , and reduce dioxygen ) . our goal with these structural analyses is to address the directing factors by which hppe converts each substrate to its final product , providing insight into the mechanisms of regiospecificity . for the mechanism of the enzyme with its natural substrate , s - hpp , much is already known , and here we provide structural insight into dioxygen binding . for r - hpp , no structural data were available , and we wished to obtain crystallographic snapshots for the stages of catalysis and compare these results to those for s - hpp . first , in considering how the fe(ii / iii ) potential is modulated in hppe to favor oxidation by dioxygen , our previous snapshots of s - hpp  hppe suggested that direct bidentate binding of the electron - donating substrate molecule s - hpp to fe(ii ) would be sufficient to activate the iron center.(15 ) we now observe that r - hpp also binds to iron in a bidentate manner via its phosphonate and hydroxyl oxygen atoms ( figure 2 ) . second , we previously considered how reactive intermediates are protected in the hppe active site when s - hpp is the substrate . we identified an induced fit mechanism in which a switch in the s - hpp binding mode from monodentate to bidentate induces a conformational change of two -strands that seals off part of the active site , protecting reactive catalytic intermediates.(15 ) interestingly , this induced fit mechanism is in play with r - hpp as well ( figure 3 ) . because the two enantiomeric substrates use the same coordination sites for binding to iron and both use the same binding pocket in the enzyme ( figures 2 and 5 ) , both substrates are able to induce the same conformational change in the -hairpin upon bidentate substrate binding . while much of the active site is buried from solvent due to this conformational change , there is a small channel available for dioxygen to enter and reach the only open coordination site on fe in the s - hpp bound hppe structures.(15 ) we have proposed that this site is the location of dioxygen binding.(15 ) here , we use no as a mimic for dioxygen binding and confirm that no does bind to this open coordination site ( figure 4 ) , providing the first structural evidence in support of this hypothesis . the binding of dioxygen to the fe(ii ) center is proposed to lead to the reversible formation of a transient fe(iii)superoxo species and facilitate hydrogen atom abstraction from s - hpp ( scheme 2a ) . in the final steps of the mechanism , the substrate radical intermediate collapses , forming the epoxide product fosfomycin , while dioxygen is fully reduced in the presence of nad(p)h , forming two water molecules ( scheme 2a ) . one water molecule may remain bound to iron until it is displaced by the binding of a second substrate molecule , consistent with the observation of multiple water molecules bound to iron in the absence of substrate.(15 ) the second water molecule would be released from iron and may occupy the farther water binding site ( 3.9  ) , trans to his180 as described above . ( a ) stereoview comparing a 2fo  fc omit map density ( contoured at 1.0  ) of s - hpp ( cyan carbons , blue mesh ) and r - hpp ( green carbons , green mesh ) from the co(ii)hppe structures . ( b ) 2fo  fc omit map density ( left ) and cartoon ( right ) for s - hpp , with accessible hydrogen atom in blue and inaccessible hydrogens in red . ( c ) 2fo  fc omit map density ( left ) and cartoon ( right ) for r - hpp . ( d ) left , superposition of r - hpp ( carbons in green ) with a hypothetical conformation ( not observed ) of r - hpp ( carbons in wheat ) in omit map density for r - hpp . right , chemical structure of r - hpp shown in a hypothetical conformation ( not observed ) shown as in ( d ) , left . accessible hydrogen in blue , and arrow indicates predicted distance . position of no from the no  s - hpp  fe(ii)hppe structure is shown in ( b)(d ) for reference . ( a ) conversion of s - hpp to fosfomycin is initiated by h - atom abstraction from c1 . ( b ) conversion of r - hpp to the keto - product probably occurs via h - atom abstraction from c2 . while this series of structures nicely explains the observed reaction of s - hpp with hppe to form an epoxide , so far this discussion has only noted similarities between s - hpp and r - hpp binding and has not described any differences that can be invoked to explain why r - hpp forms a ketone rather than an epoxide product . our structural data suggest that the formation of different products is not due to the use of differential binding sites on the enzyme , but rather it is due to the conformation that the substrate itself must assume such that it fits well into the same binding site in the enzyme . for r - hpp to bind in a bidentate mode to fe , utilizing the same binding pocket on the enzyme , adjustments must be made , but here it is the substrate rather than the enzyme that adjusts . r - hpp adopts an extended and staggered conformation that places the phosphonate moiety as far as possible from the methyl group ( figure 6 , also see supporting information figure 2 ) . the result of this conformational difference is that the c2 hydrogen is now closer to the dioxygen binding site than either c1 hydrogen , providing a structural rather than a chemical rationale for the observed regiospecificity in hppe catalysis . these findings are consistent with the fluoride analogue data,(22 ) which suggest that abstraction of a c1 hydrogen is involved in epoxide formation from s - hpp , but not in ketone formation from r - hpp ( scheme 1 ) . for s - hpp , abstraction of the c1 pro - r hydrogen by the iron superoxo intermediate ( scheme 2a ) would yield a substrate radical centered at c1 , which could collapse into the epoxide product . for r - hpp , hydrogen atom abstraction from c2 would lead to a c2-centered radical intermediate , which could collapse into the ketone product , 2-oxo - propylphosphonic acid ( scheme 2b ) . this series of structures has allowed us to probe the mechanism of regiospecificity for hppe , a fascinating variant in the nonheme mononuclear iron family of enzymes . we now have structural evidence for the manner in which both the s- and the r - enantiomers of the substrate bind , as well as for the location of dioxygen binding to fe . this first three - dimensional visualization of enantiomeric substrate recognition provides new insight into the stereospecificity afforded by a mononuclear nonheme iron enzyme .
hydroxypropylphosphonic acid epoxidase ( hppe ) is an unusual mononuclear iron enzyme that uses dioxygen to catalyze the oxidative epoxidation of ( s)-2-hydroxypropylphosphonic acid ( s - hpp ) in the biosynthesis of the antibiotic fosfomycin . 
 additionally , the enzyme converts the r - enantiomer of the substrate ( r - hpp ) to 2-oxo - propylphosphonic acid . to probe the mechanism of hppe regiospecificity , 
 we determined three x - ray structures : r - hpp with inert cobalt - containing enzyme ( co(ii)hppe ) at 2.1  resolution ; r - hpp with active iron - containing enzyme ( fe(ii)hppe ) at 3.0  resolution ; and s - hpp 
 fe(ii)hppe in complex with dioxygen mimic no at 2.9  resolution . 
 these structures , along with previously determined structures of s - hpp 
 hppe , identify the dioxygen binding site on iron and elegantly illustrate how hppe is able to recognize both substrate enantiomers to catalyze two completely distinct reactions .