denitrification is an important process 
 in biology that involves the sequential reduction of nitrate ( no3 ) to nitrite ( no2 ) , nitric oxide ( no ) , nitrous oxide ( n2o ) , and finally 
 to dinitrogen ( n2 ) , carried out by several different metalloenzymes .
 n2o + h2o ) is a key 
 step of this process and is catalyzed by nitric oxide reductases ( nors ) .
no is an important molecule in biology because 
 it impacts events ranging from blood pressure regulation , neurotransmission , and immune response in mammalian cells 
 to transcriptional regulation and biofilm 
 formation in bacteria .
the presence of nors in pathogenic 
 bacteria such as pseudomonas aeruginosa helps to 
 detoxify no and allow the bacteria to survive .
furthermore , an increase in n2o production caused 
 by the use of artificial fertilizers generated from artificial nitrogen 
 fixation has disrupted the global nitrogen cycle , as well as highlighted 
 n2o s potent ability to deplete ozone . despite the biochemical , biomedical , and environmental significance 
 of nors , structural features responsible for its activity and a clear 
 mechanistic understanding of its reaction , particularly the membrane - bound 
 nors from bacteria , are not well understood .
bacterial nor is a complex enzyme consisting of a c - type heme , 
 a heme b , and a heme b3/nonheme iron ( feb ) center .
electrons are delivered from 
 heme c to heme b and then to the heme b3/feb active site , where no is reduced to n2o ( figure 1 ) .
the active site of nor consists of 
 a high - spin ( hs ) heme b3 and feb coordinated by three histidine and one glutamate residues ( figure 1 ) .
three mechanisms of no reduction by nors have been proposed 
 ( scheme 1 ) .
briefly , the trans mechanism suggests that both 
 heme b3 and feb sites bind 
 no , one each , before n 
n bond formation , while in the cis heme b3 mechanism , a second no electrophilically attacks 
 a heme - bound no . in the cis feb mechanism ,
representation of the 
 electron - transfer pathway from cnor ( left ) and the active site structure 
 including feb ( orange sphere ) pdb 3o0r ( right ) .
enzymatic and mechanistic studies 
 of native bacterial nors are complicated by the presence of several metal sites ( three hemes 
 on a nonheme iron ; see figure 1 ) , which makes spectroscopic studies difficult , as well as 
 difficulties in purifying the protein in high yield and homogeneity 
 because nors are membrane proteins .
synthetic 
 models of nor have been used to complement the study of the native 
 enzyme to great success .
the recent progress made in synthetic modeling has been summarized 
 by other articles in this special forum and will not be duplicated 
 here . to complement both native 
 enzyme and synthetic modeling approaches ,
we have used small , stable , 
 easy - to - purify , and well - characterized proteins such as myoglobin 
 ( mb ) as  scaffolds  to make biosynthetic models of more 
 complex metalloenzymes . while a great deal of effort has been put 
 forth to understand both the structure and function of native enzymes 
 and their variants using biochemical and biophysical methods , an ultimate test of our knowledge of this class of enzymes is creating 
 functional models that mimic both the structure and function of native 
 enzymes .
in contrast to studying native 
 proteins in a  top - down  approach , which can identify necessary structural features responsible for function , biosynthetic modeling is a  bottom - up  approach that 
 elucidates structural features sufficient for activity . 

furthermore , the biosynthetic models may be amenable to investigation 
 in ways that have not yet been developed for the native enzymes ( e.g. , 
 replacement of the heme in the active site with a non - native cofactor , 
 such as zinc(ii ) protoporphyrin ix ( ppix ) ) . on the other hand
, thanks to recent progress in molecular biology 
 and protein biochemistry , protein models can now be more readily prepared 
 than by the chemical synthesis of models of complex metalloenzymes 
 such as nor because the latter method requires rigorous synthetic 
 skills . for example , it normally takes about 1 week to construct , 
 express , and purify protein models with a yield of 100 mg / l 
 of escherichia coli culture ; it would take much longer 
 to prepare heme feb models chemically with lower yield 
 because the synthesis of porphyrin - containing models is quite challenging 
 and requires multistep synthesis . despite challenges associated with 
 preparing synthetic analogues of complex enzymes ,
remarkable progress 
 has been made to understand the structure / function of complex enzymes 
 using synthetic models .
furthermore , it is becoming clear that noncovalent , secondary coordination 
 sphere interactions around the primary coordination sphere , such as 
 hydrophobicity and hydrogen - bonding interactions , often involving 
 structurally well - defined water , can play a key role in enzymatic 
 function . addressing these issues in 
 modeling
requires a rigid framework that allows the introduction of 
 these elements at specific locations .
biosynthetic modeling is an 
 ideal choice in addressing this issue because such secondary coordination 
 sphere interactions can be conveniently introduced at a specific location 
 of the rigid protein scaffold , without elaborate synthesis of model 
 compounds that may be more flexible .
therefore , although biosynthetically 
 designed protein models are intermediate in complexity compared to 
 the complex native proteins and synthetic analogues , they contain 
 many features of both the proteins and small - molecule models , providing 
 us with unique constructs to understand the structure , function , and 
 mechanism of complex metalloenzymes . in this article we will use the following nomenclature 
 convention to designate various biosynthetic models and their corresponding 
 metallated and nitrosylated derivatives .
febmb1 and febmb2 represents the first and second generations of biosynthetic 
 model proteins , respectively .
the corresponding metallated derivatives 
 are represented as m - febmb1(fe ) where the m represents a metal ion with the designated 
 oxidation state ( ii ) occupies the nonheme feb center and 
 the fe represents fe - protoporphyrin ix ( heme ) 
 in the heme - binding site . when the feb center is empty , 
 it will be represented as e - febmb1(fe ) .
when 
 the fe - protoporphyrin ix ( heme ) is replaced with zn - protoporphyrin ix , it will be designated as m - febmb1(zn ) . based on this convention , fe - febmb1(fe ) and fe - febmb1(fe )
the corresponding nitrosyl derivatives , 
 feno - febmb1(zn ) 
 and fe - febmb1(feno ) , indicate no binding to the nonheme feb center and heme fe center , respectively . in order to complement studies of native nors 
 and its synthetic models ,
our group utilizes small , easy - to - purify , 
 and well - characterized proteins like mb as the scaffold to prepare 
 biosynthetic models of nors .
this endeavor 
 was built upon our initial success in using mb to prepare structural 
 and functional models of heme copper oxidases ( hcos ) by introducing 
 a cub center in the distal pocket of sperm whale myoglobin 
 ( swmb ) through l29h / f43h mutations ( called cubmb ) . because nors and hcos belong to the same superfamily with similar 
 overall structural folds , and some hcos
have been shown to exhibit 
 nor activity , we first investigated the cross reactivity of this 
 hco mimic cubmb to reduce no and found that the presence 
 of cu at the cub site in cubmb indeed 
 displayed nor activity with consumption of 2 mol of no / mol of cubmb / min , similar to that of hco from thermus thermophilus ( 3 mol of no / mol of hco / min ) .
encouraged by the above success , we turned our attention to 
 mimic both the structure and function of native nors . at the time 
 of our pursuit , there was no crystal structure of nor available to 
 guide the rational design of a nor model using mb .
however , biochemical 
 studies and sequence homology analysis have indicated that , in addition 
 to the presence of fe in the feb center ( instead 
 of cu in the cub center ) , nors contain at least 
 two conserved glu residues in the active site that are absent in the 
 hcos . because cubmb did not bind fe and thus did not show any nor activity in the absence of 
 a metal ion in the nonheme metal center , we decided to introduce a 
 glu to the cubmb .
after evaluation of several positions 
 to introduce the glu through computer modeling and energy minimization , 
 we found the best candidate to be v68e , called e - febmb1(fe ) ( l29h / f43h / v68e swmb ) .
this protein binds fe readily ( figure 2a ) and the metal bound fe - febmb1(fe ) displays nor activity , making it the first structural and functional 
 model of nor .
overlays of cnor ( yellow ) 
 with ( a ) fe - febmb1(fe ) ( cyan ) , 
 ( b ) fe - febmb2(fe ) ( green ) , and 
 ( c ) fe - febmb1(zn ) ( magenta ) . 

feb sites are shown as brown spheres and amino acid residues 
 as sticks , and the water molecule involved in hydrogen bonding is 
 shown as a cyan sphere in part b. because there are at least two conserved glu in the active 
 site of nors , we decided to investigate the role of the second glu 
 in the biosynthetic models . while there is no room to introduce the 
 second glu in the primary coordination sphere of the feb center
, we evaluated introducing the second glu in the secondary 
 coordination sphere of the feb center .
we found i107e to 
 be at an ideal location to provide an extended hydrogen - bonding network 
 around the feb center ; thus , a 
second - generation  
 model ( i107e - febmb1(fe ) , called e - febmb2(fe ) was prepared , which also bound fe(ii ) in the 
 feb site and the metallated derivative fe - febmb2(fe ) improved the nor activity over fe - febmb1(fe ) by nearly 100% .
both fe - febmb1(fe ) and fe - febmb2(fe ) were prepared before 
 the publication of the first crystal structure of cytochrome c dependent nor ( cnor ) .
after 
 the crystal structure of cnor became available , we overlaid the crystal 
 structures of fe - febmb1(fe ) and 
 fe - febmb2(fe ) with that of native 
 nor ( figures 2a , b ) 
 and were pleased to see that , in addition to displaying nor activity , 
 both biosynthetic models mimic native cnor structurally .
this accomplishment , achieved through computational 
 modeling guided by homology modeling with structurally related proteins 
 and by activity that mimics those of native enzymes , demonstrated 
 the immense potential of biosynthetic approach in making close structural 
 and functional models of native enzymes .
spectroscopic studies of fe - febmb1(fe ) and fe - febmb2(fe ) using fourier transform infrared ( ftir ) and resonance raman 
 ( rr ) have shown that heme - bound no adopts a strong nitroxyl character 
 through interactions with the nonheme iron , and time - resolved rapid - mixing experiments provided evidence for 
 both heme and nonheme nitrosyl complexes , supporting the trans mechanism .
additionally , electron paramagnetic resonance 
 ( epr ) studies of fe - febmb2(fe ) reacted with excess no showed the formation of a five - coordinate 
 low - spin ( 5cls ) ferrous heme species due to cleavage of the proximal 
 histidine bond .
epr measurements taken 
 below 30 k of fe - febmb1(fe ) and 
 fe - febmb2(fe ) upon the addition 
 of 1 equiv of no show signals at g = 6.1 , which likely 
 arise from exchange coupling of an s = /2 6cls { feno } heme and s = 2 nonheme fe .
heme nitrosyl species have been spectroscopically probed , but a more complete understanding of the nonheme nitrosyl was limited 
 to only a few studies because the spectroscopic 
 signals of the heme often dominate the spectra of nors over the nonheme 
 iron center , hampering our understanding of the role of nonheme iron 
 in nor reactivity .
recently , we have replaced the native heme ( iron 
 protoporphyrin ix , feppix ) in fe - febmb1(fe ) with znppix , which also bound fe(ii ) , and the fe - febmb1(zn ) derivative allowed for a thorough 
 spectroscopic and computational investigation into the feb nitrosyl complex selectively , without interference from the heme 
 nitrosyl . by using uv  vis absorbance , epr , and mssbauer 
 spectroscopies , as well as x - ray crystallography ( figure 2c ) and density functional theory 
 ( dft ) calculations , the nonheme nitrosyl was characterized as an s = /2 { feno } complex , 
 using the enemark  feltham notation , best described as a hs ferrous iron antiferromagnetically coupled 
 to an no radical . to further probe the interaction of our nor 
 model with no ,
we report here uv  vis and nuclear resonance 
 vibrational spectroscopy ( nrvs ) measurements . using this information
, 
 we can systematically characterize the intermediates illustrated in scheme 1 because they are 
 likely to have distinctly different spectroscopic signatures .
nrvs 
 gives a complete and quantitative vibrational frequency spectrum for fe - enriched nuclei .
it offers a selectivity similar to that 
 of rr spectroscopy but is not bound by the optical selection rules 
 of rr or ir spectroscopy .
this is especially 
 important in the study of nors , given that the fe  no stretching 
 mode vibrations are ir - silent and decompose upon laser irradiation .
unfortunately , performing nrvs requires extremely 
 concentrated protein samples ( > 5 mm ) that are impractical when 
 working with native enzymes such as nor .
however , studies have already 
 been performed on mb and its mutants , thus offering 
 our nor mimics a unique opportunity to utilize this advanced spectroscopic 
 technique that would otherwise be inaccessible to native proteins .
vis spectroscopy was 
 used to monitor no binding to fe - febmb1(fe ) and zn - febmb1(fe ) during nrvs sample preparation .
representative 
 spectra are shown in figure 3 . upon the addition of 1 equiv of no to fe - febmb1(fe ) , where the nonheme site 
 was reconstituted with fe , the soret peak at 433 nm ( figure 3 , black curve ) underwent 
 a blue shift to 419 nm ( figure 3 , red curve ) , corresponding to the formation of a 6cls { feno } species and another broad peak at 398 nm corresponding to 
 a 5cls { feno } species ( figure 3 , red curve and inset ) , and this assignment 
 is confirmed by nrvs results ( vide infra ) .
the addition of 1 equiv 
 of no to the zn - febmb1(fe ) sample also caused a blue shift of the soret peak from 
 434 nm ( figure 3 , cyan 
 curve ) to 403 nm ( figure 3 , purple curve ) , corresponding to the formation of a 5cls 
 { feno } species .
vis spectra of fe - febmb1(fe ) ( black curve ) , zn - febmb1(fe ) ( cyan curve ) , and 
 the corresponding mononitrosyl derivatives fe - febmb1(feno ) ( red curve ) and zn - febmb1(feno ) ( purple 
 curve ) .
the inset shows deconvolution of the soret region of fe - febmb1(feno ) demonstrating 
 two components : one peak at 419 nm corresponding to the 6cls { feno } species and a second broader peak at 398 nm corresponding 
 to the 5cls { feno } species .
the presence of both 6cls 
 and 5cls { feno } species is consistent with the nrvs results 
 ( vide infra ) . in the case of the zn - febmb1(feno ) sample ,
the presence of the soret peak 
 at 403 nm is also consistent with the presence of 5cls { feno } also observed by nrvs ( vide infra ) .
nrvs exploits technology developed at third - generation synchrotron 
 light sources to monitor the vibrational properties of mssbauer 
 nuclei , including fe . tuning 
 of a monochromatic x - ray beam in the vicinity of the nuclear resonance 
 reveals vibrational sidebands displaced from the recoilless resonance 
 observed in conventional mssbauer spectroscopy .
a growing 
 number of nrvs applications exploit its exclusive and quantitative 
 sensitivity to vibrational motions of the probe nucleus .
specifically , each vibrational mode contributes to the measured 
 signal in direct proportion to the mean - squared displacement of the 
 probe nucleus along the beam direction , and well - established data 
 analysis methods directly extract a partial 
 vibrational density of states ( vdos ) for that measurement . for 
 a randomly oriented ensemble of molecules containing fe
, each vibrational normal mode contributes to the fe vdos an area equal to the fraction efe2 of the mode s kinetic energy 
 associated with motion of the fe nucleus .
the information 
 content of the vdos is quantitative , allowing direct 
 comparison with vibrational predictions on an absolute scale .
the vdos is comprehensive because all vibrations 
 involving fe contribute , without the artificial restrictions 
 imposed by selection rules in more familiar vibrational spectroscopies 
 ( ir and raman ) . finally , the vdos is uniquely site - selective because only motion of the fe will contribute , even 
 in a macromolecule containing thousands of other atoms . on the 
 basis of these characteristics , nrvs is a uniquely valuable probe 
 of protein active sites containing iron .
several investigations have 
 reported the vibrational dynamics of iron in heme proteins and iron porphyrins using nrvs .
nrvs measurements on oriented single 
 crystals of iron porphyrins exploit the sensitivity to motion along 
 the direction of the x - ray beam to provide additional insights into 
 the interpretation of the results on heme proteins .
proteins with nonheme iron sites are equally amenable to 
 nrvs investigations , which have informed the structural 
 characterization of reaction intermediates in nonheme iron enzymes .
vibrational spectra resulting from previous 
 nrvs measurements on proteins containing multiple iron atoms contain 
 superposed contributions from all sites . here
, we demonstrate for the first time that we can specifically 
 label either the heme or nonheme iron sites of fe - febmb1(fe ) with fe , allowing us to 
 independently monitor vibrations of iron at each site . partial unfolding 
 of the protein at low ph allows removal of the heme and reconstitution 
 of the protein with fe - enriched ppix , following the same 
 procedure as that used for previous nrvs investigations on native 
 mb .
heme vibrations will dominate the 
 nrvs signal of the reconstituted protein , even after the incorporation 
 of natural abundance iron ( or another metal ) into the nonheme site 
 because the natural abundance of fe is only 2% .
similarly , 
 the incorporation of fe into the nonheme site of e - febmb1(fe ) reconstituted with natural abundance 
 heme should allow us to specifically monitor the vibrations of the 
 nonheme iron .
the vdos determined from nrvs measurements on 
 reduced fe - febmb1(fe ) ( figure 4 ) demonstrate that 
 specific labeling of the heme and nonheme iron sites with fe allows us to distinguish vibrations at distinct sites within the 
 same protein .
the vibrational signal from the heme iron strongly resembles 
 that reported for native mb .
the dominant 
 feature of the vdos includes contributions from vibrations of the 
 axial fe 
nhis bond to his 93 and of the equatorial 
 fe  npyr bonds to the four heme pyrrole nitrogen 
 atoms at approximately 230 and 250 cm , respectively . 

the fe  nhis frequency is well - known from rr measurements 
 on heme proteins , where this vibration is strongly enhanced upon excitation 
 into the soret band .
the nrvs signal is determined by the relative 
 amplitude of iron motion and also includes the fe  npyr vibrations , which are not easily observable using other spectroscopies .
site - selective 
 enrichment of fe - febmb1(fe ) with fe allows independent monitoring of iron vibrations at either 
 the heme or nonheme site ( fe - febmb1(fe ) ) .
the upper and lower traces present the partial 
 vdos of the heme and nonheme iron of the reduced proteins , respectively , 
 derived from such measurements and reflect the distinct coordination 
 of iron at each site .
the heme vdos is nearly identical with that 
 reported for reduced native mb from horse 
 heart , where contributions from the fe  nhis vibration 
 perpendicular to the heme and vibrations of the in - plane fe  npyr bonds to the heme pyrrole nitrogen atoms were identified .
although individual vibrations are not resolved 
 for the less symmetric nonheme site , the stiffness derived from the 
 vdos ( table 1 ) , nevertheless , 
 reflects the lower coordination of iron in this environment . here 
 and in subsequent figures ,
error bars reflect experimental uncertainties 
 determined from counting statistics , while solid traces represent 
 a five - point running average of the experimental vdos .
the nonheme iron of reduced fe - febmb1(fe ) displays a clearly distinct 
 vibrational signal dominated by a broad feature with a peak near 230 
 cm .
the crystallographic model includes his 29 , 
 his 43 , his 64 , glu 68 , and a water molecule as ligands to the nonheme 
 iron .
the relatively featureless signal 
 observed for the nonheme iron apparently masks the vibrational structure 
 that one might expect in light of this diverse ligand field , and the 
 reduced symmetry in comparison with the heme site may further increase 
 the vibrational complexity .
we identified multiple unresolved vibrational 
 modes contributing to a similar broad vibrational feature in reduced 
 cytochrome c , based on a quantitative comparison 
 with fe / fe isotope shifts observed in rr 
 measurements , and attributed this complexity to the reduced symmetry 
 of iron coordination in the distorted heme .
conformational heterogeneity is well - documented for native mb and 
 may also broaden vibrational features .
regardless of the reasons , 
 the unresolved vibrational complexity may hinder the identification 
 of well - defined iron - ligand vibrations .
fortunately , the vibrational 
 information revealed by the nrvs measurement yields a quantitative 
 measure of the coordination strength even in the absence of detailed 
 vibrational frequency assignments .
the vdos d( ) 
 determines the stiffness,1an effective force constant that directly 
 measures the force required to displace the iron with its coordination 
 environment held fixed .
the stiffness 
 for both sites is much lower than that for the ls heme iron in reduced 
 cytochrome c , where 
 the stiffness was more than 300 pn / pm .
the stiffness is consistent 
 with the presence of a hs iron at both sites in reduced fe - febmb1(fe ) and fe - febmb1(fe ) .
the stiffness of the heme iron in reduced fe - febmb1(fe ) is the same as that in native mb 
 ( table 1 ) , confirming 
 the expectation that the introduction of the nonheme metal site does 
 not significantly affect the coordination strength of the heme iron . 

however , the stiffness of the nonheme iron environment is significantly 
 lower than that determined for the heme iron .
the slightly lower force 
 restraining the iron in the nonheme site presumably reflects its reduced 
 coordination .
similar conclusions follow from data 
 recorded on oxidized fe - febmb1(fe ) ( figure 5 ) .
a feature near 270 cm dominates the 
 heme iron vdos , which strongly resembles that previously reported 
 for native swmb .
because iron ligand 
 vibrations are undoubtedly the primary contribution to this feature , 
 this indicates that the ligation of the heme iron , to his 93 and to 
 a neutral water molecule , is the same in fe - febmb1(fe ) as it is in native mb .
in particular , 
 these data provide no indication for an oxo group bridging the two 
 iron sites , as observed for the oxidized 
 state of nor ( scheme 1 ) , and the hs nrvs signal from the heme iron contrasts with the ls 
 heme iron reported for cnor from ps .
the iron vdos of oxidized fe - febmb1(fe ) , shown in the upper trace , strongly resembles 
 that reported for native mb from sperm 
 whale , indicating that coordination of the heme iron is unaffected 
 by the presence of the additional nonheme iron engineered in the distal 
 pocket . in spite of the limited signal , the nonheme iron vdos in fe - febmb1(fe ) ( lower 
 trace )
clearly reports vibrations from a distinct iron site characterized 
 by a reduced coordination strength . as seen above for reduced fe - febmb1(fe ) , the vdos for the nonheme iron is clearly 
 distinct from that for the heme iron , in spite of a relatively low fe concentration and a consequently reduced signal in the fe - febmb1(fe ) sample . 

this dual confirmation of successful site - specific labeling of each 
 site illustrates the opportunity to probe the reactivity of each iron 
 independently .
the iron vdos determines an additional averaged 
 force constant , the resilience2which provides information distinct from the stiffness . as defined 
 more generally by zaccai , the resilience3measures the 
 rate at which the mean - squared displacement xfe of the probe atom ( here , iron ) 
 increases with temperature .
nrvs lacks the energy resolution to capture 
 highly anharmonic motions that contribute to temperature - dependent 
 measurements of xfe using techniques such as inelastic neutron scattering or 
 mssbauer spectroscopy above a
 dynamical transition  
 near 200 k. on the other hand , at temperatures below 200 k , we have 
 shown quantitative agreement between determinations of xfe from mssbauer 
 measurements on oxidized cytochrome c at a series 
 of temperatures and the values expected 
 on the basis of the iron vdos determined using nrvs at a single temperature .
the vibrational contribution to the resilience 
 ( eq 2 ) captures this 
 temperature variation in a single parameter , with lower values of 
 the resilience characterizing environments with large fluctuations 
 of the fe probe atom .
the resilience spectrum ( eq 4 ) suppresses contributions from 
 localized iron ligand vibrations and highlights low - frequency oscillations 
 of the protein that drive translational motion of both iron sites 
 in reduced fe - febmb1(fe ) and fe - febmb1(fe ) . quantitative agreement between the areas determined for both sites 
 yields values for the resilience that are identical , within experimental 
 uncertainty ( table 1 ) .
nevertheless , comparison as a function of frequency reveals subtle 
 differences in the coupling of long - range protein fluctuations to 
 these two sites .
low - frequency vibrations 
 play the primary role in determining the resilience , as we illustrate 
 by directly plotting the integrand4 in eq 2 as a  resilience spectrum  in figure 6 .
the resilience is equal to 
 the inverse of the area of this spectrum and is primarily determined 
 by vibrations below 100 cm .
molecular dynamics 
 simulations on mb and cytochrome c show similar mean - squared 
 displacements for all heme atoms including iron , supporting our suggestion that 
 translation of the heme in response to fluctuations of the embedding 
 protein matrix make the primary contribution to the nrvs signal below 
 100 cm . as a result
, we interpret the resilience 
 as a measure of the elastic properties of the protein environment . 

previously , we found a significant increase of the resilience in cytochrome c in comparison with mb . here
, 
 we find that the resiliences of reduced fe - febmb1(fe ) and native mb are the same , within 
 experimental uncertainty , suggesting that the introduction of the 
 additional nonheme metal site does not seriously perturb the elastic 
 properties of the protein .
moreover , small differences in the coupling 
 of the protein fluctuations to the heme and nonheme iron sites , apparent 
 from examination of figure 6 , average out to yield values for the resilience that agree 
 quantitatively for the two distinct sites , within the experimental 
 uncertainty .
this further supports the notion that the resilience 
 quantifies global properties of the embedding protein and contrasts 
 with the sensitivity of the stiffness to differences in the coordination 
 of the two iron sites . in short
outer - sphere  force constant that 
 probes the elasticity of the embedding protein , in contrast with the 
 probe of the immediate coordination environment , as quantified by 
 the stiffness .
the vibrational dynamics of the heme iron undergo 
 noticeable changes upon exposure to no .
the vibrational signal from 
 fe - febmb1(feno ) 
 containing fe - enriched hemes covers a wider frequency 
 range than that in the absence of no , with significant features resolved 
 beyond 500 cm ( figure 7 ) .
the experimentally determined stiffness 
 for zn - febmb1(feno ) exceeds 300 pn / pm ( table 1 ) , indicating a substantial increase in the coordination forces 
 exerted on the iron .
we observed stiffnesses exceeding 300 pn / pm for the 
 ls heme iron in reduced cytochrome c. the heme iron vdos reveals that the presence of a second 
 metal in the nonheme site influences no binding to fe - febmb1(fe ) and zn - febmb1(fe ) .
no stretching 
 vibrations , clearly resolved above 400 cm , probe 
 the axial ligation . for reference , dashed lines indicate fe  no 
 stretching frequencies reported for native horse heart mbno ( 452 cm ) , characteristic of 
 a six - coordinate complex with no coordinated trans to a histidine 
 ligand , and for fe(dpix)(no ) ( 528 cm ) , a typical five - coordinate heme no complex . a 
 substantial fraction of hemes exhibit an fe  no stretching frequency 
 characteristic of five - coordinate heme nitrosyls when either zn or fe is present in the nonheme site .
this 
 contrasts with previous measurements on native mbno , which revealed a nrvs signal consistent with six - coordinate 
 heme no .
the presence of well - resolved 
 features yields more specific information on individual fe 
ligand 
 bonds . in particular , previous nrvs measurements have identified an 
 fe  no stretching mode in the 520530 cm range in five - coordinate heme no complexes ,
binding of an imidazole ligand trans to no weakens the fe  no 
 bond , and we observe this mode at lower frequencies 
 in the 450460 cm range for these six - coordinate 
 heme no complexes .
one well - characterized six - coordinate 
 heme no complex is native mbno , where this fe  no vibration 
 appears at 452 cm and contributes to both the 
 rr and nrvs signals . unlike native mbno , fe  no 
 stretching frequencies near 530 cm characteristic 
 of five - coordinate heme no contribute to the experimental vdos of 
 fe - febmb1(feno ) 
 and zn - febmb1(feno ) upon the introduction of a divalent metal in the nonheme site 
 ( figure 7 ) .
this result 
 supports previous evidence for formation of a five - coordinate heme 
 no complex in fe - febmb1(feno ) 
 exposed to excess no , which was based on observation of the same 
 fe  no vibration at 522 cm using rr spectroscopy 
 as well as the presence of a 1660 cm n  o 
 stretching frequency in ir measurements .
the altered heme ligation in response to the neighboring nonheme 
 metal contrasts with the insensitivity of the unligated heme to the 
 nonheme metal noted above ( figures 4 and 5 ) and demonstrates that 
 the nonheme metal specifically influences the structure of the heme 
 ligand complex . in the presence of 1 equiv of no , the fe - febmb1(feno )
vdos 
 ( figure 7 ) also has 
 a feature with a 480 cm frequency that we attribute 
 to six - coordinate heme no .
because the nrvs signal depends only on 
 the mean - squared vibrational amplitude of the iron and on the relative 
 population of contributing species , the fe - febmb1(feno ) vdos suggest comparable amounts 
 of five- and six - coordinate heme no ( figure 7 ) .
interestingly , the fe  no frequency 
 is significantly increased with respect to that observed for native 
 mbno , providing additional information on how 
 the nonheme metal influences the electronic structure of the neighboring 
 heme no complex .
the contribution of a vibrational signal attributable 
 to six - coordinate heme no is significantly reduced for zn - febmb1(feno ) in the presence 
 of 1 equiv of no .
560 cm in six - coordinate heme no complexes . experimental characterization of its kinetic energy distribution 
 based on isotope shifts indicates that this vibrational mode primarily 
 involved motion of the central nitrogen atom of the feno unit . on this basis
, this n - centered 
 vibration can be qualitatively described as an feno bending mode to 
 distinguish it from the fe  no stretching mode that contributes 
 more strongly to the nrvs signal .
however , it must be emphasized that 
 neither mode can exhibit pure feno bending or fe  no stretching 
 character for the nonlinear feno unit . both modes exhibit rather modest 
 soret enhancement in raman scattering from six - coordinate heme no 
 complexes , but
the feno bending frequency is more reliably detected 
 in heme proteins because of its large sensitivity to n / n substitution and is thus more often reported .
although 
 the iron amplitude and thus the nrvs signal is necessarily smaller 
 for the feno bending vibration , the fe - febmb1(feno ) vdos includes minor features 
 near 380 and 580 cm consistent with contributions 
 from the feno bending vibration of five- and six - coordinate heme no 
 complexes , respectively , supporting conclusions based on the stronger 
 fe  no stretching frequency discussed above .
raman and 
 ir measurements on fe - febmb1(feno ) resulting from reaction with stoichiometric no also identify 
 feno bending and n  o stretching frequencies that are 1520 
 cm higher and 7080 cm lower , respectively , than those typically observed for six - coordinate 
 heme no .
together , the unusual values 
 for all three vibrations of the feno fragment suggest that the nonheme 
 fe strongly perturbs the electronic structure of heme 
 no .
in particular , it is conceivable that the fe cation 
 electrostatically predisposes the heme - bound no to the electron transfer 
 that will ultimately be required for reactivity . as found above
, the 
 nonheme iron influences the coordination of the heme , strengthening 
 the fe  no bond and weakening the fe 
in contrast , the vibrational dynamics of the nonheme iron 
 in fe - febmb1(fe ) 
 exposed to 1 equiv of no do not differ significantly from those observed 
 in the absence of no ( figure 8) .
this indicates that the ligation and electronic structure 
 of the nonheme iron is insensitive to the structural and electronic 
 changes that take place upon no binding to the heme iron .
moreover , 
 it indicates that the nonheme iron has a much lower affinity for no 
 than the heme iron does .
the vdos of the nonheme iron atom reveals perturbations 
 in fe - febmb1(zn ) 
 when the nonheme site is saturated with excess no ( see materials and methods for details of sample preparation ) . 

when fe is present in the heme site , on the other hand , 
 the nonheme iron vdos exhibits no significant change upon reaction 
 with stoichiometric no , in contrast with the clear signatures for 
 no binding to the heme iron seen in figure 7 .
replacement of the heme iron with znppix eliminates the possibility 
 of no binding to the heme and allows the investigation of no binding 
 to the nonheme iron selectively .
the vdos of the nonheme iron in the 
 resulting fe - febmb1(zn ) ( figure 8) in the 
 absence of no strongly resembles that observed for fe - febmb1(fe ) under the same conditions 
 ( figure 4 ) , indicating 
 that the structure of the nonheme iron site is insensitive to the 
 substitution of the heme metal . however , noticeable changes in the 
 nonheme vdos of fe - febmb1(zn ) take place in the presence of excess no ( figure 9 ) , which we attribute to the 
 binding of no to the nonheme iron forming the feno - febmb1(zn ) under these conditions
. computational 
 models for the no - ligated nonheme feb site in feno - febmb1(zn ) using differing 
 functionals yield quantitative predictions for the iron vdos .
comparison 
 with the experimental vdos for feno - febmb1(zn ) in the presence of excess no is consistent 
 with a substantial contribution from no - ligated iron .
the red trace 
 indicates the contribution from iron motion along the fe  no 
 bond direction and highlights the variability of the predicted fe  no 
 stretching frequency , which shifts from 376 cm using b3lyp to 454 cm using m06l .
one significant advantage of the nrvs method is the relative 
 ease of quantitative comparison with dft predictions .
overall , such comparisons provide 
 useful guidance for interpreting experimental results , but we have 
 found that predicted vibrational frequencies for the feno fragment 
 exhibit significant dependence on the functional used for dft calculations . for five - coordinate heme no complexes , 
 predicted fe  no stretching frequencies vary by nearly 200 cm .
dft investigations of nonheme feno complexes also reveal significant 
 variability of the electronic structure predicted using different 
 functionals .
it remains to be established 
 whether any currently available functional adequately accounts for 
 electron correlation in iron nitrosyl complexes .
examination 
 of a wide variety of functionals found that m06l gave the best overall account of the iron vdos for the 
 five - coordinate nitrosyl heme complex fe(oep)(no ) .
the vdos predicted using this functional is presented both 
 as the lower trace in figure 9 and , for comparison , as a filled area behind the experimental 
 vdos in the upper trace of figure 9 .
the m06l prediction does exhibit significant correspondence 
 with the experimental signal , supporting the conclusion that direct 
 no ligation accounts for the observed vibrational changes .
unfortunately , 
 because of the limited fe concentration , the relatively 
 low signal level from this sample precludes an experimental identification 
 of the fe  no stretching frequency .
the biosynthetic models have allowed 
 us to provide insight into nors that is otherwise difficult to obtain 
 in studying native enzymes .
for instance , to the best of our knowledge , 
 the nonheme feb in native nors has not been replaced or 
 removed , making it difficult to assess the role of feb in 
 the activity of nors .
in contrast , because the biosynthetic models 
 are purified without a nonheme metal ion , investigations into the 
 role of iron or other nonheme metals is greatly simplified by changing 
 the nonheme metal source that is used ( e.g. , fecl2 vs zncl2 ) . therefore , our biosynthetic model allowed us to answer 
 the previously unaddressed question of what would happen if feb was replaced with cub .
given the structural homology 
 between hcos and nors and their known cross reactivity , a fascinating 
 issue arises as to the role of each class of enzymes different 
 nonheme metal .
activity assays using iron or copper as the nonheme metal ion both 
 demonstrated nor activity , while controls using redox - inactive zinc 
 did not possess nor activity .
this study 
 demonstrated a critical insight that a redox - active metal ion was 
 needed to confer nor activity , an insight that would 
 not be possible when studying the native enzymes .
in addition , 
 we also demonstrated that the glutamate ligand to feb is 
 essential for both iron binding at the nonheme site and nor activity .
finally , an extended hydrogen - bonding network 
 was shown as a critical component in improved nor activity in our 
 biosynthetic models when a glutamate residue ( i107e ) , was introduced 
 at the secondary coordination sphere into our model to facilitate 
 proton transfer to the active site .
the ultimate goal of studying native enzymes and their models is 
 to unravel the details of how they work and apply that understanding 
 to other related enzymes as well as biomedical and biotechnological 
 applications .
this goal can best be achieved by thorough mechanistic 
 characterization , which has been achieved to great success in our 
 biosynthetic models of nors .
for example , although progress has been 
 made in elucidating the structural aspects of nors aided by recent 
 success in solving the x - ray structure of cnor , understanding the 
 mechanism of the enzyme continues to be problematic due to several 
 technical barriers . to illustrate , even though the isolated enzyme 
 cnor is reactive to no with a moderate turnover rate under steady - state 
 conditions , in the reduced form the enzyme shows very slow turnover 
 in pre - steady - state conditions because of the presence of an obscure 
 structural form of the enzyme .
flash - flow experiments with the carbonyl 
 complex of cnor can result in fast reaction kinetics .
 vis 
 changes of the protein , which is dominated by the signals from the 
 high - affinity heme site and does not provide any information on the 
 events occurring at the feb site .
apart from these 
 experimental challenges , the presence of multiple configurations of 
 the oxidized cnor further hinders our understanding of the mechanistic 
 aspects .
in one such configuration where the enzyme exists as a -oxodiferric 
 complex , strong magnetic coupling between the five - coordinate heme 
 fe ( his is dissociated from heme iron in this form ) 
 and nonheme feb is observed , while in other cases , only weak magnetic coupling was observed .
in addition , there are no experimentally accessible methods to selectively 
 probe the no complex of the feb site because the high - affinity 
 heme site dominates spectroscopic signatures including uv  vis , 
 epr , mssbauer , and nrvs . owing to these practical problems , 

n bond formation from cleavage 
 of the n  o bond ( scheme 1 ) . in the first route ,
, one no molecule binds each of the heme iron and 
 nonheme feb sites in a trans configuration , where both 
 iron centers are present as { feno } complexes .
this step 
 is followed by the reductive activation of the dinitrosyl moiety , 
 leading to the formation of a hyponitrite dianion intermediate , where 
 both iron centers are now oxidized to fe . in the  cis 
 heme b3 mechanism  , supported by theoretical studies , the first no binds to the heme 
 fe , followed by reductive activation of the no complex , 
 which is stabilized by electrostatic interactions with feb .
next , a second no electrophilically attacks the first 
 heme - bound no , leading to the formation of a hyponitrite dianion , 
 which is electrostatically stabilized by feb . finally , in the  cis feb mechanism  , both 
 no units bind to the feb site and the hyponitrite dianion 
 form is stabilized by electrostatic interactions with the heme fe . in all of these proposed mechanisms ,
it is also unclear 
 how the dianion leads to the product formation , e.g. , whether this 
 hyponitrite intermediate becomes protonated , followed by chemical 
 rearrangement of the complex , is also not well understood . with 
 these hurdles in understanding the mechanism of nor using native enzymes , 
 simpler protein - based model systems that are stable , easy - to - prepare , 
 and well - characterized are needed . to this end ,
engineered e - febmb1(fe ) and e - febmb2(fe ) and their corresponding metallated and nitrosyl derivatives have 
 provided the much needed insight into the mechanistic aspects of nors , 
 as summarized below .
resonance raman studies have shown that 
 in the reduced form both these models exist as 5chs heme in both the 
 absence and presence of fe in the feb site . however , in the presence of 1 equiv of no , both 
 proteins loaded with the feb site form stable 6cls { feno } complexes at the heme site .
one important revelation from 
 these studies was the presence of exceptionally low (no ) stretching 
 and high (feno ) frequencies compared to all reported 6cls heme 
 nitrosyl complexes .
these results were attributed to ferric heme iron(iii ) 
 nitroxyl ( feno ) complex , where no was stabilized by electrostatic interactions with 
 the feb site .
strong back - donation from heme iron caused 
 an increase in the (feno ) frequency , while the negative charge 
 on no resulted in a decrease in the (no ) frequency .
in the 
 event of excess no addition , both proteins form a [ { feno}]2 trans nitrosyl dimer , leading to n2o formation , 
 supporting the so - called  trans mechanism  . under single - turnover conditions , using ftir 
 studies , no n2o production was observed in febmb1 , suggesting that the presence of the feb site is not 
 enough to reduce no to n2o .
however , in fe - febmb2(fe ) , 50% n2o production 
 was observed , suggesting that the presence of the second glutamate 
 is critical for n2o formation , presumably by facilitating 
 proton transfer via a hydrogen - bonding network during turnover .
unproductive 
 complexes in both the proteins are characterized by a trans dinitrosyl 
 complex , where the heme iron is present as a 5cls { feno } species with a dissociated heme  his bond and a second no 
 bound to the feb site .
surprisingly , from stopped - flow 
 and rapid freeze quench experiments , no binding to the feb site was observed to be kinetically favored with a t1/2 of 1 ms , followed by binding of the second 
 no to the heme iron , leading to the trans dinitrosyl 6cls { feno}-febno complex .
this finding provided experimental 
 evidence that feb binds no before it is bound to heme b3 , which was suggested previously , but not confirmed , 
 in a study of ps .
nautica nor . in feno - febmb1(feno ) , the dinitrosyl complex leads to the formation of a dead - end 
 5cls { feno } species , where the heme  his bond is 
 dissociated , but in feno - febmb2(feno ) , the presence of the second glutamate residue leads to 50% 
 effective turnover , which results in a decreased rate of dissociation 
 of the proximal heme  his bond , leading to the formation of 
 the dead - end complex .
during the decay of the trans dinitrosyl complex 
 ( 6cls heme { feno}/feb - no ) , the hyponitrite 
 intermediate was not observed in either protein derivative , in contrast 
 to proposed mechanisms .
furthermore , when excess no was added after 
 formation of the 6cls { feno } complex , the same dinitrosyl 
 complexes of feno - febmb1(feno ) 
 and feno - febmb2(feno ) were observed 
 ( vide supra ) , ruling out the formation of any electrophilically attached 
 second no .
this observation , therefore , does not support the so - called 
  cis heme b3  , as proposed 
 by theoretical studies . as stated above , a major barrier to 
 understanding the nor mechanism is the difficulty associated with 
 isolating pure feb - no complexes due to the presence of 
 a high - affinity heme site . to circumvent this critical methodological 
 barrier in native nors , in a recent effort we spectroscopically probed 
 no binding to the feb site after replacing the high - affinity 
 heme with znppix .
such a strategy can not 
 be easily applied with native nors since the heme can not be selectively 
 replaced because of the complex nature of the enzyme . from epr ,
mssbauer , 
 and quantum mechanics / molecular mechanics calculations on the no derivative 
 of feno - febmb1(zn ) , the electronic 
 state of feb - no could be best described as hs s = /2 fe - no having a high ferrous character and a radical nature on no .
the 
 radical nature on no would promote n  n bond formation by radical 
 coupling with a heme - bound no and thus would support the trans mechanism . 

these results highlight the usefulness of biosynthetic models of complex 
 enzymes within easy - to - produce and well - characterized proteins . taken 
 together , the engineered nor models have provided important insights 
 into the reaction mechanism of nor and support the proposed  trans 
 mechanism  of no reduction by nors .
results presented here exploit the ability to replace metals at either 
 site , illustrating an important opportunity enabled by the biosynthetic 
 approach .
selective substitution with fe provides an 
 independent structural probe for either of the two metal sites in 
 fe - febmb1(fe ) .
nrvs measurements 
 quantify the forces exerted on fe by its coordination 
 environment and indicate the presence of hs iron at both sites in 
 the absence of substrate .
reduced and oxidized proteins serve to model 
 the initial and final states , respectively , in scheme 1 , although the vibrational signals provide 
 no evidence for a solvent - derived ligand bridging the metals in the 
 oxidized state .
rather , vibrations of the heme iron are comparable 
 to those reported for native mb , confirming that the heme is unperturbed 
 by the engineered nonheme site . on the other hand
, the observation 
 of distinct vibrational dynamics for the nonheme iron confirms successful 
 site - specific labeling with fe .
substitution of 
 a redox - inactive zn ion for fe allows the 
 preparation of stable mononitrosylated intermediates that would precede 
 the formation of the putative ( and unstable ) dinitrosylated intermediates 
 depicted in scheme 1 .
the presence of zn in the nonheme site perturbs the 
 vibrational properties of the adjacent heme no complex , in a manner 
 consistent with electron transfer to the no ligand .
this suggests 
 that the electrostatic influence of the nonheme fe in 
 nors could act to promote the enzymatic reaction in either the trans 
 or cis heme mechanism . with zn in the heme site ,
alteration 
 of the nonheme iron vibrations upon exposure to no confirms that no 
 can bind to the nonheme fe if the heme site is unavailable , 
 as would be required in the trans mechanism .
considerable advances 
 in the biosynthetic modeling of nors have been achieved recently , 
 and given that both the resting state and ligand - bound 
 and reduced forms of cnor have been crystallized , our understanding of nor modeling should only improve moving 
 forward .
importantly , models that can perform enzymatic turnover will 
 be critical because we are unaware of any model , biosynthetic or otherwise , 
 that is capable of reducing no with turnover numbers comparable to 
 those of the native enzymes . in conjunction with this , new models 
 that more closely replicate the secondary coordination sphere of native 
 nors must be developed because these interactions are critical for 
 improving activity . with improved structural information 
 into the active site of nor , fine - tuning of factors such as the heme 
the creation of such models will 
 provide further insights into the reaction mechanism and activity 
 of nors .
all samples 
 were prepared in a 50 mm bis - tris ph 7.3 buffer after chelexing overnight , 
 followed by ph adjustment and filtration to remove chelex beads .
buffers 
 were degassed in a schlenk line for 5 h by several cycles 
 of freeze  pump  thaw prior to their transfer into an 
 anaerobic chamber ( coy laboratories , inc . ) for sample preparations . 

dry sephadex g25 beads ( ge healthcare ) were suspended in a buffer 
 solution and degassed in the schlenk line for several hours before 
 transfer into the glovebag .
all solid materials were kept under vacuum 
 overnight in the antechamber prior to transfer into the glovebag . 

all protein solutions were exchanged from a 100 mm phosphate ph 7 
 buffer to a 50 mm bis - tris ph 7.3 buffer outside the glovebag using 
 small size - exclusion columns ( pd 10 columns , ge healthcare ) preequilibrated 
 in an exchangeable buffer .
the protein solutions were then degassed 
 by three cycles of freeze  pump  thaw in a schlenk line 
 and brought into the glovebag .
a diethylamine nonoate ( dea - nonoate ; 
 250  nm = 6.5 mm cm ; cayman chemicals ) solution prepared in 10 mm naoh 
 and used as the no source was degassed by three cycles of freeze  pump  thaw 
 before transfer into the glovebag .
a stock solution of fecl2 was prepared inside the glovebag by dissolving solid fecl2 in degassed water .
e - febmb1(fe ) was purified using a known 
 protocol , as reported previously .
a similar 
 protocol was employed for e - febmb1(fe ) purification except that in this case fe - labeled 
 heme ( frontier scientific ) was used during the protein refolding step . 

the identity of each of the purified proteins was confirmed by electrospray 
 ionization mass spectrometry .
the r / z of pure protein was > 4 in a 100 mm potassium phosphate ph 7 buffer . 

molar extinction coefficients 
 406 = 175 mm cm for met e - febmb1(fe ) , 433 = 143 mm cm for deoxy e - febmb1(fe ) , and 427 of 136.2 mm cm for e - febmb1(zn ) were used to determine the concentrations of the corresponding 
 proteins .
a total of 300 ml of deionized 
 water and 1 ml of 9.14% methanolic hcl ( 285.6 l of 32% hcl 
 + 714.4 l of methanol ) were degassed and transferred into the 
 glovebag .
a total of 25 mg of fe metal ( 0.44 mmol ; cambridge 
 isotope lab ) was taken into a small dry nmr tube and transferred into 
 the glovebag .
the degassed water was transferred into a small water 
 bath and heated to 60 c using a hot plate equipped with 
 a stir bar .
the nmr tube containing fe was immersed into 
 the water bath , and 350 l of 9.14% methanolic hcl ( 0.88 mmol ) 
 was added to the tube .
the reaction was allowed to proceed for 34 h until the gas 
 evolution ceased .
the schlenk flask was removed from 
 the bag , immersed in a dry ice / ethanol slush bath , and slowly opened 
 to vacuum in a schlenk line .
the flask was then slowly warmed to 100 
 c using a water bath while in vacuum .
after the solvent evaporated 
 and the solid turned from green to white , the water bath was replaced 
 with an oil bath and heated to 160 c , allowing the residual 
 methanol to evaporate .
the product was cooled to room temperature 
 slowly , purged with argon , sealed , and weighed .
nrvs data were collected at the advanced photon source on beamline 
 3id - d , as described in detail elsewhere .
briefly , the x - ray energy was scanned in the vicinity of the fe nuclear resonance at 14.4125 kev in steps of 0.25 mev . 

data were recorded on frozen solutions at 510 mm protein concentration 
 at temperatures of 6080 k , and each measurement was the average 
 of 1535 scans .
a comparison of early and late scans confirmed 
 the absence of spectroscopic changes during x - ray exposure .
met e - febmb1(fe ) was reduced inside the glovebag with excess dithionite 
 and passed through a small hand - packed size - exclusion column ( sephadex 
 g25 ) preequilibrated with a 50 mm bis - tris ph 7.3 buffer to remove 
 excess dithionite and eluted with the same buffer .
the eluted protein 
 was then concentrated to 1 mm ( 433  nm = 143 
 mm cm ) , and the nonheme site 
 was reconstituted with 1.0 equiv of either fecl2 or zncl2 ( prepared freshly in the inert - atmosphere bag ) 
 added in aliquots of 0.25 equiv with 15 min between each addition . 

e - febmb1(zn ) was transferred into the glovebag 
 after degassing and concentrated to 1 mm ( 427  nm = 136.2 mm cm ) , and the 
 feb site was reconstituted with fe without reducing the protein because znppix is redox - inactive .
reconstitution 
 of the feb site with fe or zn , as desired , was ensured by checking the uv  vis spectrum 
 after metal addition , as evidenced by shifting of the soret peak from 
 427 nm in e - febmb1(zn ) and 433 nm in e - febmb1(fe ) , in the absence of the nonheme metal 
 to 429 nm in feii - febmb1(zn ) 
 and 434 nm in fe - febmb1(fe ) or fe - febmb1(fe ) in the reconstituted protein , respectively .
when applicable , 
 oxidation of the heme iron and nonheme iron was achieved after reconstitution 
 by the addition of excess ferricyanide and then passage through a 
 small size - exclusion column .
the feb reconstituted proteins 
 were then concentrated to 1015 mm before loading 15 
 l of the samples into the well of a high - density polyethylene 
 block sample holder inside the glovebag , transferred outside , and 
 frozen immediately where other components ( sapphire window , copper 
 block , brass screws ) were assembled .
nitrosyl derivatives were 
 prepared using the following protocol . for e - febmb1(fe ) , 1 equiv of no was added to the fe or zn reconstituted proteins present at 1 mm concentration . 
 at each step
, 0.25 equiv of no was added to the reconstituted proteins 
 in the form of dea - nonoate , allowing enough time to for no release 
 between each addition ( t1/2 = 16 min at 
 ph 7.3 ) .
no binding to the proteins was confirmed by measuring uv  vis 
 spectra of no - bound samples .
similarly , for fe reconstituted fe - febmb1(zn ) , the protein was kept at 1 mm concentration before no addition . 

excess no was added , in the form of dea - nonoate as described above , 
 to fe - febmb1(zn ) 
 to saturate the feb site with no .
after no binding , the 
 samples were further purified by another pd10 column equilibrated 
 in a 50 mm bis - tris ph 7.3 buffer to remove any trace impurities including 
 the decay product of dea - nonoate .
all of the nitrosyl complexes thus 
 prepared were then concentrated to 1015 mm and loaded 
 into nrvs cells described above .
the protocol of adding no at 1 mm 
 of the reconstituted proteins followed by concentrating to higher 
 concentrations has proven to be a successful strategy in our studies , 
 as we have recently reported .
an aliquot 
 of each of the above concentrated samples was diluted , and their uv  vis 
 spectra were checked inside the glovebag to ensure that no changes 
 in no coordination occurred during the final step of sample preparation .
the vtz basis 
 set was used for the iron orbitals and 
 6 - 31 g * for all other atoms .
the computational model for the nitrosyl 
 complex of the nonheme site of feno - febmb1(zn ) used the atomic coordinates deposited 
 in the protein data bank under access code 3k9z(39 ) for his 
 29 , his 43 , his 64 , glu 68 ( with -carbon atoms replaced by 
 terminal methyl groups ) , the iron and a ligated water , and added no 
 as a sixth ligand .
energy optimization of the experimentally observed s = /2 state yielded nearly octahedral 
 coordination for the iron , with an fe 
n  o angle varying 
 from 142.8 ( b3lyp ) to 143.2 ( m06l ) .
the atomic displacements 
 of the vibrational normal modes were used to calculate the iron vdos 
 as described previously .
 this forum article focuses on recent 
 advances in structural and spectroscopic studies of biosynthetic models 
 of nitric oxide reductases ( nors ) . 
 nors are complex metalloenzymes 
 found in the denitrification pathway of earth s nitrogen cycle 
 where they catalyze the proton - dependent two - electron reduction of 
 nitric oxide ( no ) to nitrous oxide ( n2o ) . while much progress 
 has been made in biochemical and biophysical studies of native nors 
 and their variants , a clear mechanistic understanding of this important 
 metalloenzyme related to its function is still elusive . 
 we report 
 herein uv  vis and nuclear resonance vibrational spectroscopy 
 ( nrvs ) studies of mononitrosylated intermediates of the nor reaction 
 of a biosynthetic model . 
 the ability to selectively substitute metals 
 at either heme or nonheme metal sites allows the introduction of independent 57fe probe atoms at either site , as well as allowing the preparation 
 of analogues of stable reaction intermediates by replacing either 
 metal with a redox inactive metal . 
 together with previous structural 
 and spectroscopic results , we summarize insights gained from studying 
 these biosynthetic models toward understanding structural features 
 responsible for the nor activity and its mechanism . 
 the outlook on 
 nor modeling is also discussed , with an emphasis on the design of 
 models capable of catalytic turnovers designed based on close mimics 
 of the secondary coordination sphere of native nors . 
Introduction
Summary and Outlook
Materials and Methods
Calculations