mammalian nitric oxide 
 synthase ( nos ) is a homodimeric flavo - hemoprotein 
 that catalyzes the oxidation of l - arginine to no , with nadph 
 and o2 acting as cosubstrates .
there are three nos isoforms : endothelial , neuronal , and inducible 
 ( enos , nnos , and inos , respectively ) .
each nos subunit contains a 
 c - terminal electron - supplying reductase domain with binding sites 
 for nadph ( the electron source ) , fad , and fmn and an n - terminal catalytic 
 heme - containing oxygenase domain .
the oxygenase and fmn domains are 
 connected by a calmodulin ( cam ) binding linker , which is tightly bound 
 to cam in inos at a basal calcium level , while cam binding to nnos 
 and enos requires a significantly higher ca concentration .
the binding of cam activates 
 no synthesis in 
 enos and nnos by ( i ) enabling the fmn domain transitions between its 
 electron - accepting position ( input state ) and an electron - donating 
 position ( output state ) and ( ii ) facilitating the proper docking of the fmn domain on to 
 the oxygenase domain , which enables efficient fmn 
cam is also important for proper 
 alignment of the fmn and heme domains in inos .
heme iet is essential for the 
 delivery 
 of electrons required for o2 activation in the heme domain 
 and the subsequent no synthesis by nos .
the crystal structures are only available for truncated nos domains , 
 including the oxygenase domains of each of the nos isoforms , rat nnos reductase constructs , and cam - bound 
 human inos fmn domain , along with cam 
 bound to peptides corresponding to the cam - binding region in enos , nnos ( pdb i d 2o60 ) , and inos ( pdb i d 3gof ) .
although high - resolution 
 x - ray crystallographic structures have been obtained for certain segments 
 of nnos and other nos isoforms , the details of how the various tethered 
 nos domains interact with cam and how cam binding influences functionally 
 important interactions among the domains are not known .
herein 
 we employed the pulsed electron paramagnetic resonance ( epr ) 
 relaxation - induced dipolar modulation enhancement ( ridme ) technique to monitor the binding of spin - labeled cam ( sl cam ) to nnos and 
 to determine the position of the nitroxide spin label ( sl ) with respect 
 to the heme centers of the dimeric oxygenase domain .
wild - type cam 
 does not contain cysteine amino acid residues , but a cysteine can 
 be readily introduced at a selected cam site by site - directed mutagenesis . 
 in this work , to enable the spin - labeling , a cysteine was introduced 
 at position 110 .
recent work showed that this mutation has little 
 effect on nnos enzyme activation . in 
 order to simplify the system ,
we used the bidomain nnos oxyfmn construct , 
 which contains the oxygenase and fmn domains , as well as the cam binding 
 region , but lacks the nadph and fad binding domains .
this nos construct is a valid model of the electron - donating ( output ) 
 state of the fmn domain .
construction , 
 purification , and spin - labeling of t110c mutant cam are described 
 in the supporting information .
the spin - labeling 
 efficiency and selectivity have been confirmed by tryptic digestion 
 and mass spectrometry ( figure s1 in the supporting 
 information ) .
the 
 rat nnos oxyfmn plasmid was cotransformed 
 with cam expression vector ( p209 ) into escherichia 
 coli bl21(de3 ) cells by electroporation .
the transformed 
 cells were grown at 37 c in terrific broth in the presence of 
 100 g / ml ampicillin and 34 g / ml chloramphenicol .
protein 
 expression was induced by adding induction cocktail ( 0.5 mm isopropyl 
 -d - thiogalactopyranoside , 0.4 mm -aminolevulinic 
 acid , and a pinch of riboflavin ) when the cell culture reached an 
 optical density of 1.0 at 600 nm .
after incubation at 25 c for 
 40 h , the cells were harvested at 4 c .
cells were resuspended 
 in the lysis buffer ( ph 7.5 ) containing 50 mm tris - hcl , 10% glycerol , 
 200 mm nacl , 0.5 mm cacl2 , 5 mm -mercaptoethanol , 
 three complete protease inhibitor tablets ( roche ) , 10 m h4b , and 0.5 mg / ml lysozyme .
the lysate was centrifuged at 30  000 rpm for 40 
 min at 4 c .
the supernatant was then loaded onto a co - chelating column ( talon metal affinity resin , clontech ) pre - equilibrated 
 with five bed - volumes of equilibration buffer ( 50 mm tris - hcl , 10% 
 glycerol , 200 mm nacl , 0.5 mm cacl2 , ph 7.5 ) .
the column 
 was washed with ten bed - volumes of equilibration buffer containing 
 15 mm imidazole , and the contents were then eluted with a 15150 
 mm imidazole gradient in the elution buffer ( 50 mm tris - hcl , 10% glycerol , 
 200 mm nacl , 0.5 mm cacl2 , ph 7.5 ) .
the eluted protein 
 was pooled , concentrated , and dialyzed into the storage buffer ( 50 
 mm tris - hcl , 10% glycerol , 200 mm nacl , 1 mm dtt , 10 m h4b , ph 7.5 ) .
for preparation of the sl cam - bound nnos 
 epr samples , the coexpressed 
 cam was removed by dialysis against the storage buffer containing 
 2 mm egta .
the molar concentration of the nnos protein was determined 
 on the basis of the heme content via difference spectra of the ferrous 
chelex 100 iron form 
 resin ( bio - rad ) was used to remove the background ca from 
 the nanopure water for preparation of the calcium - free epr buffer 
 ( 50 mm bis - tris propane , 200 mm nacl , 3 mm imidazole , 42% ethylene 
 glycol , ph 7.4 ) .
imidazole was added to convert the heme centers of 
 the nnos oxyfmn construct to the low - spin fe(iii ) state .
the spin - labeled 
 t110c cam and nnos oxyfmn were concentrated and dialyzed into the 
 epr buffer and the epr buffer containing 20 m h4b , respectively .
solutions containing sl cam and nnos oxyfmn were 
 gently mixed ( final concentration 110 m and 55 m , respectively ) , 
 and cacl2 was then added to prepare solutions with final 
 total ca concentrations of 0.22 , 0.5 , 1 , 2 , and 5 mm . 

about 40 l of each solution was transferred into epr tubes 
 and rapidly frozen in a pentane and liquid nitrogen slurry .
the experiments have been performed 
 on the homemade broad - band pulsed epr spectrometer at the university 
 of arizona .
the overall structural model 
 of this work is shown in figure 1 . according 
 to this model , without ca present in solution
, cam does 
 not form a complex with nnos ( panel a of figure 1 ) . as the ca concentration increases , cam binds ca ( up to four ca ions per cam molecule ) , and 
 cacam can now bind to the cam binding region spanning 
 residues 725745 of the flexible tether connecting the oxygenase 
 and fmn domains of nnos ( panels b and c in figure 1 ) .
( a ) without ca in solution , no 
 cam binding to nnos occurs , and the docking complex between the fmn 
 domain and the heme domain does not form .
( b and c ) when ca ions are present in solution ( up to four ca ions per 
 cam molecule ) , cam binds to the nnos cam - binding motif , which facilitates 
 the formation of the iet - competent docking complex between the fmn 
 and heme domains .
the cam - bound nnos is in equilibrium between the 
 structurally disordered open state ( b ) and the iet - capable docked 
 state ( c ) . for the purposes of this work
, 
 we will distinguish two qualitatively 
 different structural states of the cam - bound nnos : the open ( undocked ) 
 state , where the bound cam can move freely within the range of positions 
 permitted by the flexibility and length of the tether ( panel b in 
 figure 1 ) , and the docked state ( panel c ) , 
 where the docking of the bound cam to the oxygenase domain facilitates 
 and strengthens the docking of the fmn domain .
the specific structural models for the docked and undocked 
 states used in the interpretation of the experimental results of this 
 work will be presented below .
the structural arrangements described 
 in the previous section can be distinguished by using ridme , one of 
 the pulsed epr techniques sensitive to the magnetic dipole interaction 
 between the sl and the fe(iii ) ions of the heme centers .
the meaningful 
 use of the alternative pulsed epr technique sensitive to the magnetic 
 dipole interaction between the paramagnetic centers , the electron 
electron 
 double resonance ( eldor ) , is hampered for this system by the unfavorable 
 epr properties of the ferric heme centers ( strong n electron 
 spin - echo envelope modulation ( eseem ) and significant g - anisotropy ) .
this has been discussed in our previous work , where 
 the distance of 18.8  between the heme center and the fmn semiquinone 
 radical of the docked fmn domain was determined by ridme .
the ridme measurements were performed 
 using the refocused stimulated electron spin - echo ( ese ) pulse sequence 
 ( figure s2 in the supporting information ) .
the refocused stimulated ese signal from the sl was measured as 
 a function of the interval between the first and second mw pulses , 
  , at a fixed time interval between the second and third pulses , t. these measurements were performed at two temperatures , tlow and thigh , selected 
 in such a way that the longitudinal relaxation time of the heme center , t1fe , was very long at tlow ( t1fe  t ) but relatively short at thigh [  
 < t1fe  t , 
 so that the time interval t effectively corresponded 
 to a complete relaxation of the fe(iii ) spins ] . the specific temperatures 
 used in our experiments were tlow = 8 
 k and thigh = 25 k. these temperatures 
 are determined by the relaxation properties of the ferric heme center(s ) 
 of nos established in our previous work . the quotient of the ese traces , vq( ) 
 = v(,thigh)/v(,tlow ) , represents 
 the ridme trace whose amplitude dependence on  is caused by 
 the magnetic dipole interaction of the sl with the fe(iii ) spins . 

an example of the original experimental ridme traces obtained in this 
 work is shown in figure s3 of the supporting information .
figure 2 shows the quotient ridme 
 traces 
 for the samples containing nnos oxyfmn and sl cam obtained at different 
 concentrations of ca and also the trace for sl cam without 
 nnos .
one can see that while the latter trace ( trace 1 ) is approximately 
 constant ( understandably so , since no fast - relaxing species is present 
 in solution ) , those of the samples with nnos represent monotonously 
 decaying functions .
the slowest decay observed for the sample with 
 [ ca ] = 0 ( trace 2 ) is caused by the magnetic dipole interaction 
 of the sl with the uniformly distributed fe(iii ) spins .
the trace 
 for the sample with [ ca ] = 0.22 mm shows somewhat faster 
 decay ( trace 3 ) , indicating the onset of cam binding to nnos .
starting 
 from [ ca ] = 0.5 mm the decay does not increase further 
 ( overlaid traces labeled as 4 in figure 2 ) , 
 which indicates the maximum concentration of nnoscam complex 
 achievable at [ ca ]  0.5 mm .
ridme traces obtained 
 by division of the refocused stimulated ese 
 decays of the sl recorded at the temperature of 25 k by those recorded 
 at 8 k. trace 1 is obtained for the sample of 110 m sl cam 
 without nnos , containing 1 mm ca .
traces 24 are 
 obtained for the samples of 110 m sl cam with 55 m nnos 
 oxyfmn construct , containing various concentrations of ca . for traces 2 and 3 , [ ca ]
trace 4 represents an overlay of the traces obtained for [ ca ] = 0.5 , 1 , 2 , and 5 mm .
experimental conditions : mw frequency , 30.276 
 ghz ; b0 = 1077.2 mt ( maximum of the sl 
 signal ) ; mw pulses , 20 , 20 , 20 , and 30 ns ; time interval between the 
 second and third mw pulses , t = 20 s .
figure 3 shows the [ ca ] dependence 
 of the ridme effect in the samples containing nnos relative to the 
 effect observed in the sample with [ ca ] = 0 .
this effect 
 was calculated by ( i ) dividing the ridme traces of figure 2 obtained for all of nnos - containing samples by 
 trace 2 ( to eliminate the effect of the uniformly distributed spins ) , 
 ( ii ) subtracting 1 , ( iii ) dividing the resulting traces by the average 
 of those obtained from traces in group 4 of figure 2 , and ( iv ) averaging the amplitude of the obtained traces 
 ( which are approximately horizontal lines ) over the time interval 
 . the horizontal axis is calibrated in the [ ca]/[cam ] units .
one can see that the ridme effect saturates at [ ca]/[cam ]  4 , which is expected because one cam molecule 
 can only bind up to four ca ions in its ef - hands .
since 
 the calcium concentrations are much higher than the physiological 
 concentrations ( nanomolar level ) , additional 
 discussion of ec50(ca ) for cam binding would 
 be susceptible to overinterpretation .
[ ca ] dependence of the ridme 
 effect in the samples 
 of nnos oxyfmn containing sl cam .
the ridme effect was calculated 
 by ( i ) dividing traces 24 of figure 2 by trace 2 ( to eliminate the effect of dipole interaction of the 
 sl with uniformly distributed fe(iii ) spins ) , ( ii ) normalizing the 
 resulting quotient traces by the average of the traces obtained from 
 group 4 in figure 2 ( i.e. , those corresponding 
 to [ ca ]  0.5 mm ) , and ( iii ) taking the average 
 value over the resulting normalized traces .
the analysis ( using 
 numerical simulations ) of the experimental ridme traces of figure 2 can ideally provide information about the structure 
 and relative statistical weights of the docked and open states of 
 the cam - bound nnos ( figure 1c , b , respectively ) . 
 as a preparation for this analysis
, one must divide the traces obtained 
 at [ ca ] > 0.5 mm by the trace obtained at [ ca ] = 0 .
this is the first operation of those used to obtain 
 the data 
 shown in figure 3 above , which eliminates the 
 effect from uniformly distributed matrix nnos .
the resulting traces 
 represent a sum of two contributions : ( i ) the generally nonconstant 
 ( decaying or oscillating ) contribution from the camnnos complexes 
 and ( ii ) the constant contribution from the free cam molecules ( if 
 any ) .
the black solid trace 1 in each panel of figure 4 shows the result of the division of the average 
 of the ridme traces corresponding to [ ca ]  0.5 
 mm ( since these traces were similar , the average was taken to increase 
 the signal - to - noise ratio ) by the trace corresponding to [ ca ] = 0 . in order to analyze this trace
, one has to take into account 
 that the oxygenase domain contains two heme fe(iii ) centers that simultaneously 
 interact with the sl . the longitudinal relaxation of these heme centers 
 during the time interval t ( between the second and 
 third mw pulses of the refocused stimulated ese sequence ) at 25 k 
 results in the spin flip probability of 0.5 for each of these centers . 

the resulting distribution of the relaxation outcomes is as follows : 
 25% of the fe(iii ) pairs do not flip ; in 50% of the fe(iii ) pairs , 
 one of the spins flips ( 25% each ) ; and in 25% of the fe(iii ) pairs , 
 both spins flip . in the last group ,
one - half of the population undergoes 
 a flip between | and | 
 states , while the other half flips between | 
 and | states .
the statistics of the fe(iii ) 
 spin flips is used to evaluate the resulting change in the local magnetic 
 field at the position of the sl and to calculate the ridme effect .
solid black trace 1 in each panel is the experimental 
 ridme effect in the samples of cam - bound nnos oxyfmn .
( a ) traces in 
 group 2 are examples of simulations based on the docking model in 
 figure 5 .
the various traces are simulated 
 for the distances between the sl and the two fe(iii ) centers of ( 36 , 
 31 )  ( long dashes ) , ( 40 , 36 )  ( short dashes ) , and ( 31 , 
 26 )  ( dots ) .
the solid cyan trace is an average over several 
 sl positions within the uncertainty range , including the above three , 
 ( 38 , 27 )  , and ( 34 , 33 )  .
the simulated traces were multiplied 
 by 0.4 to provide for convenient scaling of the figure .
( b ) long - dashed 
 trace 2 is simulated using the open state model in figure 6 .
short - dashed trace 3 is obtained from trace 2 
 by multiplication by 0.45 to approximately equalize its slope with 
 that of the experimental trace .
( c ) an example of simulation ( solid 
 cyan trace ) with a superposition model corresponding to 12% of the 
 docked state and 88% of the open state .
traces 2 and 3 show the contributions 
 of the open and docked states , respectively , into the cyan trace . 

see the text for details . the experimental ridme trace of figure 4 can not be explained by a single structure with a fixed distance 
 between the sl and the heme centers because in this case oscillations 
 rather than a monotonous decay would be observed ( relevant examples 
 are shown by the dashed and dotted traces in figure 4a and discussed below ) .
this indicates that the majority of 
 the cam - bound nnos is in the open state ( figure 1b ) that is characterized by a broad distribution of the sl positions 
 relative to the oxygenase domain .
the lack of well - defined oscillations 
 also prevents one from obtaining accurate information on the cam position 
 in the docked state directly from the ridme trace using the standard 
 approach , which consists of estimating the characteristic distance 
 from the oscillation frequency .
in this situation , we have to rely 
 on the structure of the docked state obtained from docking calculations 
 for validating the results of numerical simulations of the ridme kinetics .
next we will describe the structural information pertaining to 
 our analysis of the experimental ridme trace of figure 4 in terms of relative populations of the docked and open states . 

the crystal structures of nnos oxygenase domain and of the cam unit 
 bound to the cam - binding region are available ( pdb i d 2g6k and 2o60 , respectively ) , 
 and the solution structures will be assumed to be the same .
although 
 the crystal structure of the docked state has not yet been determined , 
 the computer modeling allows one to predict the relative arrangement 
 of the oxygenase and fmn domains and the bound cam unit in the docked 
 state .
we constructed a docking model of nnos fmn domain along with 
 cam on to the oxygenase domain ( figure 5 ) . 

this was accomplished by closely referencing to the docking model 
 reported recently for the system of murine inos based on hydrogen  deuterium 
 exchange mass spectrometry results . according 
 to our model
, the sl in the docked state is located at the distances 
 of about 36 and 31  ( as measured to the o atom 
 of thr110 , which corresponds in the mutant cam to s of cys110 , to which the sl is attached ) from the iron ions of the 
 two heme centers of the oxygenase domain , the distance between which 
 is 34  .
docking model of nnos fmn domain ( purple , pdb i d 1tll ) onto the dimeric 
 oxygenase domain ( cyan and green , pdb i d 4jsh ) in the presence of cam ( gray , pdb i d 2o60 ) . for clarity
the sl site 
 ( res110 ) in cam is labeled , so are the terminal residues in oxygenase 
 and fmn domains that connect with the cam - binding peptide .
the docking 
 model was constructed by carefully cross - checking against the inos 
 model reported recently in terms of which 
 residues are involved in the docking surface among the proteins . in the open state ,
the sl position 
 is assumed to be uniformly distributed 
 within a sphere of the radius corresponding to the full extension 
 of the flexible tether joining the oxygenase domain with the bound 
 cam , with the exclusion of inaccessible regions , in particular , the 
 part of space occupied by the oxygenase domain ( also see below ) .
the flexible tether joining 
 the oxygenase domain with the bound cam is a random coil spanning 
 residues 706731 . with 3.53.6  per residue
, this 
 corresponds to the maximum backbone extension of 89  1  . 

the sl is located at about 15  from phe731 , which makes the 
 total maximum possible distance between the oxygenase domain and the 
 sl equal to 104  .
the 
 sl position is averaged over the sphere centered at the peptide nitrogen 
 of tyr706 , with the radius of 104  .
the regions occupied by 
 the oxygenase domain and the gray part of the sphere on the far side 
 of the oxygenase domain ( 40  from the center to the cutoff plane ) 
 are excluded from the calculation as inaccessible .
the size of the dimeric 
 oxygenase domain has characteristic dimensions 
 of 90  50  50  and is comparable 
 with the tether length .
therefore , the bound cam can not reach the 
 parts of the spherical region on the far side of oxygenase domain . 
 predicting the exact shape of this inaccessible region
requires calculating 
 all possible configurations of the tether wrapping around the oxygenase 
 domain surface .
since the oxygenase domain shape is not accurately 
 described by any analytical function ( e.g. , a sphere or an ellipsoid ) , 
 such a calculation is prohibitively time - consuming and practically 
 unrealistic .
an approximate exclusion region , however , can be predicted 
 on the basis of comparison of the characteristic size of the oxygenase 
 domain with the tether length . in this particular case
it represents 
 a part of the sphere on the far side of the oxygenase domain shown 
 by the gray area in figure 6 .
since 
 the specific conformation of the sl or distribution thereof in cam 
 is unknown ( which is a common situation in site - directed spin - labeling ) , 
 one has to consider the uncertainty in the position of the > n  o radical fragment arising from the rotational degrees 
 of freedom of single bonds between the > n  o fragment and the s  s bridge , which is approximately 6  
 in every direction ( see figure s4 of the supporting 
 information ) .
this uncertainty is not important for the open 
 state of nos because the distances there are distributed within very 
 large limits ( 100  in every direction ) .
for the docked 
 state , however , this uncertainty has to be considered explicitly ( see 
 below ) . using the structural models presented above , we performed 
 numerical 
 simulations of the ridme effect for the docked and open states of 
 nnos . in these simulations ,
such an approximation is unavoidable 
 because the orientations of the heme g - frames with 
 respect to the oxygenase domain of nnos are not known . neglecting 
 the actual g - anisotropy [ ( g1 , g2 , g3 ) = ( 1.85 , 2.30 , 2.52 ) ] , however
, does not result in the loss of 
 potentially obtainable structural information because ( i ) the anisotropy 
 is fairly small , ( ii ) in the open state the heme 
sl radius 
 vectors are orientationally disordered with respect to the g - frames , and ( iii ) in the docked state the potential changes 
 in the ridme frequencies resulting from taking the g - anisotropy into account are smaller than the changes from the uncertainty 
 in the sl position .
the results of the calculations for the 
 docked state are shown 
 by group 2 of dashed and dotted traces in figure 4a .
these traces were calculated for three different sl positions 
 within the 6  range of uncertainty mentioned above . note 
 that they exhibit well - defined oscillations with the frequencies equal 
 to the dipole interactions ( expressed in frequency units ) between 
 the sl and the heme centers and are very different from the experimental 
 trace 1 .
a distribution of the sl position within the uncertainty 
 range does not create a better agreement : while the oscillations are 
 suppressed , the trace practically flattens out starting from 250 to 
 300 ns ( solid trace of cyan color in figure 4a ) .
the long - dashed trace 2 in figure 4b shows 
 the calculation result for the open state .
this trace exhibits significantly 
 smaller curvature and greater slope than the experimental one ( trace 
 1 ) . although the slope of the calculated trace can be reduced by assuming 
 less than 100% formation of the low - spin ferric state of the heme 
 centers or the presence of some unbound cam ( short - dashed trace 3 
 in figure 4b ) , this does not help with improving 
 the agreement because the curvature then becomes even smaller .
while the docked or open state models by themselves result in simulated 
 ridme traces significantly different from the experimental one , the 
 agreement can be reached for a combination of the two states . to estimate 
 the relative contributions of the docked and open states , the calculated 
 ridme trace for the open state ( long - dashed trace in figure 4b ) was multiplied by the scaling factor copen < 1 ( as exemplified by the short - dashed 
 trace in figure 4b ) to approximate the asymptotic 
 slope of the experimental trace and subtracted from the latter .
numerical 
 simulations of the difference trace were then performed for various 
 sl positions . the sl position in these simulations
was characterized 
 by the distance rsl from the midpoint 
 between the fe(iii ) ions of the heme centers ( that are 34  apart ) 
 and by the angle sl that the radius - vector rsl makes with the line joining the 
 heme centers ( the definitions are shown in the graphical form in figure 
 s6 of the supporting information ) . in order 
 to fit the amplitude of the difference
ridme trace , the simulated 
 traces were multiplied by the scaling factor cdock < 1 .
the simulations have shown that there is 
 only a relatively narrow 
 range of the scaling factors copen ( 0.450.52 ) 
 that result in the difference traces that could be reproduced by numerical 
 simulations .
using the smaller copen of 
 0.45 allows one to obtain the approximate fit of the difference trace 
 with sl  60 , rsl uniformly distributed between 31 and 46  , and cdock  0.1 .
rsl uniformly distributed between 
 30 and 36  , and cdock  0.075 
 ( see figures s7 and s8 in the supporting information for simulation examples ) . for comparison , the docking model ( figure 5 ) places s of cys110 ( to which 
 the sl is attached ) of mutant cam at sl  
 80 and rsl = 29  .
thus , the 
 sl position at sl  75 used in the 
 simulation is in good agreement with the docking model : the 5 
 angular difference at the distance of 30  translates 
 into the difference in position of <3  , within the uncertainty 
 range related to the sl conformational flexibility ( 6  ; 
 see figure s4 of the supporting information ) .
the solutions corresponding to the smaller sl are progressively less likely ( e.g. , at sl = 60 
 and rsl = 30  the distance between 
 the sl > n  o fragment and the predicted 
 anchoring 
 position at s is about 10  ) . using the 
 obtained values of scaling factors copen and cdock
, the population 
 of the docked state can be estimated as pdock = cdock/(cdock + copen ) .
the population of the open 
 state is popen = 1  pdock . on the basis of the range of copen ( 0.450.52 ) and the corresponding range of cdock ( 0.10.075 )
, one can estimate pdock = 15  3% , with the smaller values 
 corresponding to the more probable solution with sl  75. the simulation for sl = 75 
 that is in reasonable agreement with the experimental ridme trace 
 and with the docking model is shown in figure 4c .
the results of the present 
 ridme work indicate that the slfe(iii ) distance 
 is highly distributed and dynamic , even in the cam - bound nnos .
this 
 is consistent with the fmn domain tethered shuttle model : cam activates nnos by enabling transitions between conformational 
 states , and the fmn domain moves back and forth to contact the ferredoxin  nadp - reductase module and the nos heme domain .
cytochrome p450 reductase and p450 bm3 ( e.g. , 
 a hinge movement of the fmn domain toward the heme domain in p450 
 bm3 was proposed ) , which derives from 
 p450 reductase and p450 bm3 structures , including 
 the crystal structure of the bm3 heme 
heme iet occurs in the docked 
 state , but the interdomain fmn  heme interactions in nos enzymes 
 are rather weak , and the docking complexes are short - lived .
oxyfmn construct is 
 therefore limited by the relatively infrequent formation of the docked 
 iet - competent complexes .
we have recently shown that the retarded 
 iet in the e546n mutant of human inos oxyfmn is not caused by an altered 
 conformation of the docked fmn  heme complex but by a lower 
 population of the iet - active conformation .
on the other hand , in the nnos holoenzyme , the electron transfer 
 to the heme center is more likely limited by passage through a conformational 
 bottleneck ( that does not exist in the oxyfmn construct ) .
it is interesting to compare the findings of our ridme study 
 with the results of fmn fluorescence lifetime investigations performed 
 in solutions at room temperature .
the populations 
 of the docked and open conformations of cam - bound nnos oxyfmn in frozen 
 solution estimated from our ridme measurements are about 15% and 85% , 
 respectively .
the corresponding populations estimated for inos oxyfmn 
 from room temperature fmn fluorescence measurements are about 2530% 
 and 7570% .
fluorescence lifetime 
 data for nnos oxyfmn construct have not been reported yet , but the 
 population breakdown obtained for the full - length nnos enzyme suggests that the nnos oxyfmn construct in terms 
 of the docked and open state populations should be similar to that 
 of inos oxyfmn .
one has to note that the docked state population estimates 
 in the literature depend on the assignment of the 
 fluorescence component with 1 ns lifetime . while there is 
 little doubt that this component is caused by the fmn 
heme 
 interaction , it is not clear if the relative arrangement of the oxygenase 
 and heme domains enabling this interaction is unique and corresponds 
 to the docking complex only .
therefore , we believe that the 2530% 
 population of the docked state derived from the fluorescence investigations represents an upper limit estimate .
another factor that makes 
 the comparison of the ridme and fluorescence 
 lifetime results not entirely straightforward is that the former pertain 
 to the nos - bound cam , while the latter pertain to fmn .
the comparison 
 is justified by the fact that fmn and cam are located in close proximity 
 ( they are separated by a linker that is only 7 residues long , compared 
 to 25 residues between cam and the oxygenase domain ) and can be approximately 
 considered as a single structural unit .
therefore , we conclude that 
 the ridme and fluorescence data are at least in a semiquantitative 
 agreement . on a more general level ,
the above comparison tests 
 a commonly 
 used paradigm that the structural distribution in a frozen solution 
 represents a 
snapshot  of the dynamic equilibrium in 
 fluid solution . while this paradigm is obviously convenient , it has 
 to be used with caution .
the relevant literature appears to be rather 
 scarce , but examples of both small and 
 large temperature- and phase - dependent 
 conformational distribution changes are available .
the joint results 
 of this ridme investigation and the fmn fluorescence studies indicate 
 that for the nos oxyfmn constructs the  snapshot  approximation 
 is at least semiquantitative .
the broad structural distribution 
 of cam - bound nnos detected by 
 ridme is in agreement with a low - resolution cryo - electron microscopy 
 ( em ) study of enos holoenzyme , which 
 indicated that a large ( although unquantified ) fraction of nos is 
 in distributed conformations even when cam is bound .
the overall conformational 
 equilibrium in cam - bound nnos was estimated in another cryo - em study , which showed that the cam - bound nnos holoenzyme 
 adopts an ensemble of open and closed conformational states , with 
 only about 15% in the closed conformation .
the closed state as used 
 in the em work represents a structural 
 category that corresponds to the nnos protein folded on itself , as 
 opposed to the extended and intermediate ( v - shaped ) states where the 
 oxygenase  fmn  reductase domain
this classification of the conformational states 
 categorizes the perceived overall shapes of the entire nnos dimer , 
 but not the relative arrangement of the specific structural parts 
 of the protein .
it is important to note that although the docking 
 complex studied 
 in our work obviously corresponds to a subset of the closed conformations 
 in the em work , the specific closed conformation 
 stabilized by cross - linking and characterized in detail is very different and is far from optimal for 
 the fmn 
heme iet since the fmn domain there is still too far 
 ( 100  ) from the hemes .
in addition , the fmn domain in 
 the three - dimensional reconstruction model is proximal to the side of substrate - access channel of the heme 
 domain ( but kinetics and mutational studies have consistently shown that the fmn domain docks to the back face 
 of the heme domain where the heme is closest to the protein surface ) . 
 further fmn domain motions ( swing and rotation ) are clearly required 
 prior to the iet .
therefore , the 15% population of the closed state 
 found in the cryo - em work does not translate directly to the population 
 of the fmn 
heme iet - competent conformation and represents , 
 at best , the upper limit estimate for the docked fmn / heme state . to 
 improve the docked state geometry and statistics estimates ,
a high - resolution 
 structure of the functional full - length nos state should be obtained .
although the docked fraction of about 15% estimated by ridme measurements 
 is relatively small , it is not negligible : its presence indicates 
 that the free energies of the docked and open states are comparable . 

the presence of the measurable population of the docked state is also 
 important from the mechanistic viewpoint because it implies a possibility 
 of minor local structural adjustments in the docked state itself to 
 reach the fmn 
finally , our results are agreeable with the notion that cam docking 
 with the oxygenase domain is necessary to facilitate the rather weak 
 interdomain fmn  heme interactions required for efficient iet , 
 which was first experimentally established in an isotope exchange 
 mass spectrometry study of murine inos oxyfmn protein .
the system investigated in this work is 
 characterized by a wide 
 distribution of distances between the paramagnetic centers , and the 
 ridme trace represents a relatively featureless monotonous decay . 
 in a recent pulsed eldor study of the 
 magnetic dipole interaction between flavin semiquinone radicals in 
 fad and fmn domains of nnos ( a system qualitatively similar to ours , 
 with two domains connected by a flexible tether ) , clear oscillations 
 in the eldor trace
were observed , indicating that a significant fraction 
 of the protein was in well - defined structural states .
unfortunately , 
 no analysis of the distributed fraction was performed in their work , 
 and the percentages allotted to each fraction were not determined .
in this work , we have studied the binding of 
 sl cam to nnos oxyfmn 
 bidomain construct using pulsed epr ( the ridme technique ) to detect 
 the specific magnetic dipole interaction between the sl and the ferric 
 heme centers of nnos .
it was found that the binding saturates at [ ca]/[cam ]  4 , which is expected from the number of 
 the calcium binding sites in cam .
the analysis of the ridme traces 
 has shown that only about 15  3% of the cam - bound nnos is in 
 the iet - competent docked state at any given time , while the remaining 
 85  3% of the protein is in the open conformations characterized 
 by a wide distribution of the distances between the bound cam and 
 the oxygenase domain .
the low population of the docked state found 
 in this ridme study indicates that the cam - controlled docking between 
 the primary functional fmn and heme domains is highly dynamic .
the 
 overall approach of this work to the analysis of the ridme data can 
 be used for obtaining information about the structural state(s ) of 
 other protein systems consisting of domains connected by a flexible 
 tether .
 the 
 binding of calmodulin ( cam ) to neuronal nitric oxide synthase 
 ( nnos ) enables formation of the output state of nnos for nitric oxide 
 production . 
 essential to nos function is the geometry and dynamics 
 of cam docking to the nos oxygenase domain , but little is known about 
 these details . in the present work , the domain docking in a cam - bound 
 oxygenase / fmn ( oxyfmn ) construct of nnos was investigated using the 
 relaxation - induced dipolar modulation enhancement ( ridme ) technique , 
 which is a pulsed electron paramagnetic resonance technique sensitive 
 to the magnetic dipole interaction between the electron spins . 
 a cysteine 
 was introduced at position 110 of cam , after which a nitroxide spin 
 label was attached at the position . 
 the ridme study of the magnetic 
 dipole interaction between the spin label and the ferric heme centers 
 in the oxygenase domain of nnos revealed that , with increasing [ ca2 + ] , the concentration of nnoscam complexes increases 
 and reaches a maximum at [ ca2+]/[cam ]  4 . 
 the ridme 
 kinetics of cam - bound nnos represented monotonous decays without well - defined 
 oscillations . 
 the analysis of these kinetics based on the structural 
 models for the open and docked states has shown that only about 15 
  3% of the cam - bound nnos is in the docked state at any given 
 time , while the remaining 85  3% of the protein is in the open 
 conformations characterized by a wide distribution of distances between 
 the bound cam and the oxygenase domain . 
 the results of this investigation 
 are consistent with a model that the ca2+cam interaction 
 causes cam docking with the oxygenase domain . 
 the low population of 
 the docked state indicates that the cam - controlled docking between 
 the fmn and heme domains is highly dynamic . 
Introduction
Materials and Methods
Results and Discussion
Conclusions