bacterial multicomponent 
 monooxygenases ( bmms ) comprise a family 
 of enzymes capable of hydroxylating or epoxidizing a wide range of 
 hydrocarbons , including the greenhouse gas methane and environmentally 
 hazardous substances such as benzene and trichloroethylene .
bmm enzymes can be grouped into four classes : the three - component 
 enzymes soluble methane monooxygenases ( smmos ) , phenol hydroxylases 
 ( phs ) , and alkene monooxygenases ( amos ) , and the four - component enzymes 
 alkene / arene monooxygenases .
all bmm enzymes 
 contain three common components : a hydroxylase , a reductase , and a 
 regulatory protein .
the hydroxylase component is a 
 multi - subunit dimeric ( 222 or 22 ) protein hosting 
 a diiron center in each -subunit .
the carboxylate - bridged diiron 
 center is the locus for o2 activation and subsequent substrate 
 hydroxylation / epoxidation .
it is similar to those 
 in the r2 subunit of ribonucleotide reductase , ferritin , stearoyl acyl carrier protein 
 9 desaturase , and the aging - related protein 
 clk1 .
the reductase component is an nadh oxidoreductase 
 with an [ fe2s2 ] cluster in the ferredoxin domain 
 ( fd ) and a flavin adenine dinucleotide ( fad ) cofactor in the fad domain , 
 responsible for the reduction of the hydroxylase diiron center .
the third component , a cofactor - less regulatory 
 protein , couples nadh consumption to product formation . a key question regarding the 
 catalytic mechanism of bmm enzymes is how component protein interactions 
 achieve the timely control of electron transfer to the diiron active 
 site , dioxygen activation , and hydrocarbon substrate oxidation . elucidating component protein binding sites on the hydroxylase 
 is required as a foundation for answering such a question .
an important 
 clue comes from early crystallographic investigations of hydroxylases 
 from the three - component system smmo and the four - component 
 toluene / o - xylene monooxygenase ( tomo ) .
both hydroxylases contain a structure of c2 symmetry with a shallow depression , termed 
 the  canyon 
the canyon region was proposed as the docking 
 site for the other component proteins , and , indeed , 
 later crystallographic studies revealed that the regulatory component 
 does occupy a portion of the canyon in hydroxylase  regulatory 
 protein complexes of ph , toluene-4-monooxygenase 
 ( t4mo , another four - component bmm ) , and , 
 very recently , smmo ( figure 1b ) .
regulatory 
 protein 
 complex of smmo ( pdb i d 4gam ) : ( a ) the hydroxylase mmoh showing the canyon , and 
 ( b ) mmoh in complex with the regulatory protein mmob .
there is another 
 mmob molecule binding to the canyon on the other side of mmoh .
mmoh 
 -subunit is colored in green , -subunit in blue , -subunit 
 in yellow , and mmob in purple .
there is no crystal structure available for the 
 hydroxylase  reductase complex of any bmm enzyme . by using the 
 zero - length cross - linker 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide 
 ( edc ) , a chemical cross - linking study of smmo isolated from methylosinus trichosporium ob3b revealed that the 
 reductase , mmor , cross - linked to the -subunit of the hydroxylase 
 mmoh , and that the regulatory component , mmob , cross - linked to the 
 -subunit .
a different result was 
 obtained , however , using smmo isolated from methylococcus 
 capsulatus ( bath ) , where either the full - length mmor or its 
 fd cross - linked to the -subunit using the same cross - linker , 
 edc .
further attempts to determine the 
 binding site by identifying cross - linked residues failed .
the two 
 identified fd cross - linking sites , glu-56 and glu-91 , cross - linked 
 to the n - terminal amino group of mmoh -subunit , which is not 
 observed in the crystal structure of mmoh owing to disorder . because the mmor binding site on mmoh 
 is obscure , it was unclear 
 how the regulatory protein and the reductase might interact in the 
 complete enzyme system . simulations of steady - state oxidase and oxygenase 
 activities of smmo as a function of component protein concentrations 
 favored a non - competitive model , whereby mmor and mmob bind at distinct 
 sites on mmoh , forming a hypothetical 
 ternary complex .
the formation of such a ternary complex was also 
 proposed in a small - angle x - ray scattering ( saxs ) study , where the 
 species formed in the presence of large excess of mmob and mmor ( 1020 
 equiv of each relative to mmoh ) was modeled as a mmoh2mmob2mmor 
 complex .
later crystallographic investigations 
 of the hydroxylase  regulatory protein complexes , however , suggested 
 that the regulatory component may block the reductase binding site , but there was no direct experimental evidence for such .
the role 
 of the regulatory protein in electron transfer from the reductase 
 to the hydroxylase diiron center is also not well understood .
a determination 
 of the reductase binding site on the hydroxylase would clarify many 
 of these questions .
accordingly , in this study we determined 
 the reductase binding 
 site on the hydroxylase of smmo isolated from methylococcus 
 capsulatus ( bath ) , by using hydrogen 
the results clearly reveal 
 that the fd of mmor indeed binds to the canyon of mmoh .
more importantly , 
 the fd shares the same binding site as the core of mmob ; it therefore 
 binds competitively with mmob to mmoh .
consistent with the shared binding site , we show that mmob 
 does not facilitate , but actually inhibits , electron transfer .
overall , 
 this work presents the first experimentally determined reductase fd 
 binding site on the hydroxylase of a bmm enzyme , and it reveals how 
 the regulatory component may control electron transfer in the catalytic 
 cycle .
5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic 
 acid ( iaedans ) was obtained from molecular probes . other chemicals 
 were purchased from sigma - aldrich and used without further purification .
the expression 
 system for mmor ferredoxin domain ( fd , residues 1107 ) was 
 prepared by mutating s108 and f109 of wild - type mmor to stop codons 
 by site - directed mutagenesis , using primers shown in table s1 .
fd was expressed and purified as described previously , except that an additional step with a monoq 
 column was employed to separate apo protein without the iron  sulfur 
 cluster from the holo protein .
the expression system for the mmob 2 - 33 
 d36c mutant was prepared by site - directed mutagenesis using mmob d36c 
 as the template ; the primers are shown in table 
 s1 .
the mmob 2 - 33 d36c protein was expressed and purified 
 following the procedure described for wild - type mmob .
a 60 pmol portion of mmoh 
 was incubated with 
 fd for a final mmoh : fd concentration ratio of 1:6 during deuterium 
 labeling . under this condition ,
> 95% of the fd binding sites on 
 mmoh 
 were saturated , based on a kd value of 
 0.9 m . all mixtures were incubated for 20 min at room temperature 
 before deuterium labeling . as a control ,
mmoh alone was incubated 
 in 50 mm phosphate buffer ( ph 7.0 ) and treated exactly the same as 
 the fd - bound protein .
deuterium exchange was initiated by dilution 
 of each sample with 15-fold 50 mm phosphate buffer ( pd 7.0 ) , 99.9% 
 d2o at room temperature . at each deuterium exchange time 

point ( 10 s , 1 min , 10 min , 60 min , 4 h , 6 h , and 8 h ) , an aliquot 
 from the exchange reaction was removed and quenched by adjusting the 
 ph to 2.5 with an equal volume of quench buffer ( 150 mm potassium 
 phosphate buffer , h2o ) . quenched samples were immediately 
 frozen on dry ice and stored at 80 c until analysis . 

several undeuterated control samples were prepared in the same way 
 as the deuterium - labeled samples and were used for validation of the 
 peptic peptides of the proteins used in the deuterium labeling experiments .
each flash - frozen sample was rapidly thawed and injected into a 
 waters nanoacquity with hdx technology ( waters corp . ) .
the protein samples were digested online using 
 a 2.1 mm  30 mm poroszyme immobilized pepsin cartridge ( applied 
 biosystems ) .
the digestion temperature was set to 15 c and the 
 digestion was performed for 30 s. the cooling chamber of the ultra - performance 
 liquid chromatography ( uplc ) system , which housed all the chromatographic 
 elements , was held at 0.0  0.1 c for the entire time of 
 the measurements .
the injected peptides were trapped and desalted 
 for 3 min at 100 l / min and then separated in 14 min by a 5% 
 to 40% acetonitrile : water gradient at 40 l / min .
the separation 
 column was a 1.0  100.0 mm acquity uplc c18 bridged ethyl hybrid 
 particles ( beh ) column ( waters corp . ) containing 1.7 m particles , 
 and the back pressure averaged 8800 psi at 0.1 c .
the average 
 amount of back - exchange using this experimental setup was 1825% , 
 based on analysis of highly deuterated peptide standards .
deuterium 
 levels were not corrected for back - exchange and are therefore reported 
 as relative ; however , all comparison 
 experiments were done under identical experimental conditions , thus 
 negating the need for a back exchange correction .
the uplc step was performed with protonated solvents , thereby 
 allowing deuterium to be replaced with hydrogen from side chains and 
 the amino / carboxyl terminus that exchange much more rapidly than amide 
 linkages .
the average error in determining the deuterium levels 
 was 0.1 da in this experimental setup , consistent with previously 
 obtained values . in order to eliminate 
 peptide carryover ,
a wash solution of 1.5 m guanidine hydrochloride , 
 0.8% formic acid , and 4% acetonitrile was injected after each run .
mass spectra were obtained with a waters xevo g2 tof instrument 
 equipped with standard electrospray ionization source ( waters corp . ) . 

the instrument configuration was the following : capillary was 3.2 
 kv , trap collision energy at 6 v , sampling cone at 35 v , source temperature 
 of 80 c , and desolvation temperature of 175 c .
mass accuracy was ensured by calibration with 
 500 fmol/l human [ glu1]-fibrinopeptide b and was less than 
 10 ppm throughout all experiments .
the mass spectra were processed 
 with the software dynamx 2.0 ( waters corp . ) by centroiding an isotopic 
 distribution corresponding to the + 2 , + 3 , or + 4 charge state of each 
 peptide .
deuteration levels were calculated by subtracting the centroid 
 of the isotopic distribution for peptide ions of undeuterated protein 
 from the centroid of the isotopic distribution for peptide ions from 
 the deuterium - labeled sample .
the resulting relative deuterium levels 
 were automatically plotted versus the exchange - in time .
identification 
 of the peptic fragments was accomplished through a combination of 
 exact mass analysis and ms using identity software ( waters 
 corp . ) .
ms was performed by a series of low  high 
 collision energies ramping from 5 to 32 v , therefore ensuring proper 
 fragmentation of all the peptic peptides eluting from the lc system .
iaedans - labeled mmob d36c and 2 - 33 d36c 
 mutants were prepared 
 following procedures described previously .
concentrations of the labeled proteins were determined by using 
 the bradford assay ( bio - rad ) .
the excitation wavelength was set to 
 336 nm and emission was monitored at 490 nm .
samples were made in 
 25 mm mops , ph 7.0 buffer ; the concentration of fluorescently labeled 
 protein was 1 m .
mmoh 
 was considered to have two non - interacting binding sites ( hsite ) .
the simulation procedures are described as follows , taking the 
 titration of fd into 1 m mmoh and 1 m iaedans - labeled 
 mmob as an example .
two equilibria were considered , eqs 1 and 2 , where [ hsite ] , [ b ] , 
 and [ fd ] are the concentrations of free mmoh binding site , free mmob , 
 and free fd ; [ hsite - b ] and [ hsite - fd ] are the 
 concentrations of bound mmob and fd ; [ hsite]total , [ b]total , and [ fd]total are the total concentrations 
 of mmoh binding site ( 2 m ) , mmob ( 1 m ) , and fd ( the 
 total amount titrated in ) .
b ) 
 was determined previously to be 0.55 m ; several kd values for the h 
fd 
 complex ( kd , h  fd ) were tested to 
 allow us to choose the one that best simulated the experimental data .
the concentrations [ b ] and [ hsite  b ] were first 
 calculated for each titration point by numerically solving the simultaneous 
 eqs 1 and 2 .
these values 
 were then used to calculate the observed fluorescence anisotropy robs ( eq 3 ) , which is the 
 sum of fluorescence anisotropy of free and bound mmob weighted by 
 their fractional fluorescence intensity,3 where rb and rhsite  b are the fluorescence 
 anisotropy of free and bound mmob , and fb and fhsite  b are the 
 fractional fluorescence intensity of free and bound mmob , respectively . 

the fb and fhsite  b parameters can be expressed in terms of 
 [ b ] , [ hsite  b ] , and the molar fluorescence intensity 
 of free and bound mmob , fb and fhsite 
b:45 substituting eqs 4 and 5 into eq 3 , the fluorescence 
 anisotropy of 
 each titration point can be calculated on the basis of [ b ] and [ hsite  b ] by solving eqs 1 and 2:6 the same simulation procedures 
 were followed for the titration 
 of fd into 1 m mmoh and 1 m iaedans - labeled mmob 2 - 33 . 
 the kd value of 2.67 m for the 
 h 
b 2 - 33 complex determined in this study was used for 
 the simulation ( figure s3 ) .
several kd values ( 0.9 , 2 , and 6 m ) for the h 
fd 
 complex ( kd , h  fd ) were tested to 
 see which one best fits the experimental data . to a mixture of 10 m 
 mmoh , 20 m mmor , and 0120 m mmob in 50 mm mops 
 buffer , ph 7.0 , was added 10 mm cross - linker edc .
the reaction was 
 incubated at room temperature for 10 min and then quenched by adding 
 an equal volume of sds loading buffer .
the reaction was analyzed by sodium dodecyl sulfate polyacrylamide 
 gel electrophoresis ( sds - page ) .
the electron transfer kinetics 
 of smmo were studied by stopped - flow optical spectroscopy at 15 c . 

the tubing and syringes of the hi - tech scientific sf-61 dx2 double - mixing 
 stopped - flow instrument were made anaerobic by first flushing and 
 then incubating with 15 mm anaerobic sodium dithionite solution for 
 3 h , followed by flushing with 25 mm anaerobic mops buffer , ph 7.0 
 right before use .
the following steps were performed inside of a glovebox 
 with an o2 level of less than 0.5 ppm . to investigate electron 
 transfer from chemically reduced reductase , 40 m degassed fd 
 or mmor
was titrated with 3 mm sodium dithionite until the absorption 
 at 405 nm no longer changed , and the resulting sample was then loaded 
 into a gastight syringe . a 20 m quantity of
mmoh , or 20 m 
 mmoh premixed with 40 m mmob in the presence or absence of 
 10 m mmor , was degassed and sealed in another gastight syringe . 
 to study electron transfer from nadh , a 40 m solution of anaerobic 
 nadh
was sealed in one syringe ; 20 m mmoh premixed with 40 
 m mmor in the presence / absence of 40 m mmob was sealed 
 in another syringe .
the syringes were then taken out from the glovebox 
 and connected to the stopped - flow instrument .
equal volumes of reagents 
 from each syringe were rapidly mixed by the stopped - flow instrument , 
 and the electron transfer kinetics were monitored by recording the 
 absorbance change at 470 or 458 nm .
data were fit by two ( when chemically 
 reduced fd or mmor was used as the electron source ) or three ( when 
 nadh was used as the electron source ) exponentials , and effective 
 electron transfer rates were calculated as weighted averages of individual 
 electron transfer rate constant .
hdx - ms is a powerful tool for 
 probing protein structure , dynamics , and the binding interface .
the rationale behind hdx - ms relies on protein backbone amide protons 
 that are in constant exchange with solvent protons , or deuteriums 
 if in deuterated solvent .
the number of exchangeable protons and their 
 rates of exchange depend on factors such as ph , temperature , chemical 
 environment , and the three - dimensional protein architecture , thus reflecting the structure and dynamics of the protein .
typically , 
 protein backbone amide protons exchange rapidly with deuterons if 
 they are involved in weak or suboptimal hydrogen bonds , reside at / near 
 the surface , or are readily accessible to the solvent ; the exchange 
 rates are slower if they are involved in strong intramolecular hydrogen 
 bonds and/or are less accessible to solvent .
protein 
 binding sites , based on the reduced solvent exposure in regions that 
 constitute the binding interface . here
mmoh is a 251.3 kda homodimer that consists of three protomer 
 subunits in each monomer :  ( 60.6 kda ) ,  ( 45.1 kda ) , 
 and  ( 19.8 kda ) . upon pepsin digestion ,
165 overlapping mmoh 
 peptic peptides were detected , covering 93.9% , 93.5% , and 96.4% of 
 the sequences of the - , - , and -subunits , respectively 
 ( figure s1 ) .
the deuterium incorporation 
 and protein dynamics were followed from 10 s up to 8 h. all the peptic 
 peptides that were followed by hdx - ms are displayed in figure s2 .
most of these peptides showed low 
 deuterium uptake even after an 8 h incubation in deuterated buffer 
 ( figure 2 ) .
the data indicate very slow dynamics 
 in most of the three subunits of the homodimer , suggesting that the 
 protein is very rigid and not undergoing breathing movements indicative 
 of a dynamic and solvent exposed structure .
amide backbone hydrogens involved in hydrogen - bonding interactions 
 in secondary structural elements such as -helices and -sheets 
 exhibit slow exchange rates .
therefore , 
 the low deuterium uptake is consistent with the high helical content 
 of mmoh .
the  
 and -subunits are more protected from exchange compared with 
 the -subunit , the peptides of which seem to indicate a more 
 accessible and dynamic structure ( figure 2 ) . summary 
 of hdx - ms data for free mmoh in solution at four time points . 

the hdx - ms data are mapped onto pdb entry 1mty , with the 
 color code indicated for deuteration times shown at the bottom of 
 each image .
the hdx - ms data are shown only on one monomer ; the second 
 monomer is represented in sand color .
we next 
 used hdx - ms to locate regions of mmoh that show differences in exchange 
 upon binding to the ferredoxin domain ( fd , residues 1107 , 
 11.8 kda ) of mmor .
the 
 two isolated domain proteins both have stable structures and retain the biochemical properties of the two domains in full - length 
 mmor .
electron transfer to the diiron(iii ) 
 centers in the hydroxylase involves first , nadh reductions of the 
 oxidized fad cofactor to its hydroquinone form , followed by the [ fe2s2 ] cluster in the fd sequentially shuttling two 
 electrons from the reduced fad cofactor to the diiron center in mmoh .
the fd protein is a smaller yet 
 competent model of mmor for studying electron transfer as well as 
 binding to mmoh . among the 165 
 detected peptic peptides of mmoh in the presence of fd ( figure s2 )
, six peptides derived from the -subunit 
 and three from the -subunit exhibited lower deuterium uptake 
 compared with samples without fd ( figures 3 and s2 ) , covering residues 7088 
 and 236255 of the -subunit , and residues 3748 
 of the -subunit .
the differences in deuterium uptake were visible 
 after 1060 min deuteration but not at earlier time 
 points , possibly because the exchange rates in the absence of fd were 
 already very slow due to stable h - bonds in the -helices . in 
 the presence of
fd the reduced solvent exposure at the binding site 
 modestly reduced the exchange rates .
the differences in deuterium 
 uptake in the presence and absence of fd were subtle even at longer 
 incubation times ( up to 8 h exchange ) , but were consistent in the 
 set of overlapping peptides .
typically , differences in deuteration 
 greater than 0.4 da but less than 1 da are considered subtle .
the 
 experiments were performed in triplicate , and the average error of 
 the measurements was 0.1 da . all other mmoh peptic peptides 
 exhibited the same deuterium uptake in the presence or absence of 
 fd ( figure s2 ) .
deuterium 
 exchange kinetics for four representative 
 peptides that showed different deuterium uptake in the presence ( blue 
 traces ) or absence ( red traces ) of fd : ( a ) residues 7081 , 
 -subunit ; ( b ) residues 8288 , -subunit ; ( c ) residues 
 237242 , -subunit ; and ( d ) residues 4047 , -subunit . 

the largest number on the y - axis represents the maximum 
 amount of deuterium that can be incorporated in each peptic peptide .
we then mapped the peptides that 
 showed decreased deuterium uptake 
 in the presence of fd onto the crystal structure of mmoh ( pdb entry 4gam ) .
these peptides 
 cluster in the canyon region at the 22 interface , representing a possible fd binding site ( figure 4a ) .
this region includes the area closest to the 
 diiron center from the protein surface , a preferred binding site for 
 the iron  sulfur cluster of the reductase in order to facilitate 
 fast electron transfer . this binding site determined by hdx - ms rationalizes 
 the seemingly conflicting results of previous chemical cross - linking 
 studies . because the binding site consists of residues from both - 
 and -subunits , mmor can cross - link to either the -subunit 
 ( for smmo isolated from methylococcus capsulatus ( bath ) ) or the -subunit ( for smmo isolated from methylosinus trichosporium ob3b ) .
it is unlikely that smmos isolated from different species have different 
 reductase binding sites .
the different cross - linking results are most 
 likely due to different distributions of carboxyl groups and amino 
 groups that are required to be in close proximity for the cross - linking 
 mediated by edc .
( a ) mmoh peptides that 
 showed 
 decreased deuterium uptake in the presence of fd are mapped onto the 
 crystal structure of mmoh ( pdb i d 4gam ) .
( c ) a closer view of the binding interface in the docked 
 model : the [ fe2s2 ] cluster of fd , mmoh residues 
 at the binding interface , and the diiron center of mmoh , viewed from 
 the top of the figure .
( d ) the crystal structure of mmoh-2mmob , showing 
 that mmob covers the fd binding site .
mmoh is colored in cyan ; binding 
 site peptides of the -subunit in orange and those of the -subunit 
 in yellow ; fd in red ; and mmob in purple . to validate the hdx - ms - determined fd binding site , we performed 
 computational docking using the cluspro server .
the structure of fd ( residues 
 198 , pdb i d 1j4q ) previously determined by solution - state nmr spectroscopy and the crystal structure of mmoh ( pdb i d 4gam ) were used as input structures , and the nmr - determined fd 
 residues involved in binding to mmoh were 
 used as docking constraints .
the results show that fd covers the hdx - ms - determined 
 binding surface ( figure 4b ) .
the -subunit 
 peptides involved in binding ( yellow region in figure 4 ) seem uncovered in the docked structure ( figure 4b ) , but upon a closer examination residues 4548 
 inside the canyon were found to be in close contact with fd .
the docking 
 result is therefore in full agreement with the hdx - ms data .
a close examination 
 of the docked mmoh  fd complex reveals 
 that the diiron active site of mmoh is approximately 14  away 
 from the [ fe2s2 ] cluster of fd , a favorable 
 distance for electron transfer .
consistent 
 with hdx - ms results , the docked model reveals that fd covers the pore 
 region of mmoh , which we previously proposed as the proton transfer 
 pathway from the mmoh surface to its diiron center .
residue e240 in the mmoh -subunit , the gating residue of 
 the pore , is situated midway between the [ fe2s2 ] cluster of fd and the diiron center of mmoh ( figure 4c ) .
previously , this residue was found to shift its conformation 
 toward the protein interior upon mmob binding , closing the pore and 
 possibly bringing in a proton to the diiron active site .
we propose that an identical conformational 
 change occurs when mmor binds to mmoh during electron transfer from 
 the fd [ fe2s2 ] cluster to the carboxylate - bridged 
 diiron center , providing the mechanism for proton - coupled electron 
 transfer .
strikingly , the fd binding site determined here overlaps 
 largely with that of mmob as previously determined by the crystal 
 structure of the mmoh2mmob complex .
in particular , this structure 
 reveals that the core of mmob docks into the canyon region , covering 
 the very same area where we now conclude fd binds , while the n - terminal 
 tail of mmob binds to an adjacent location on mmoh , on the surface 
 of helices h and 4 of the -subunit , adopting a ring - like conformation 
 ( figure 4d ) .
this 
 observation requires the fd of mmor and the core of mmob compete for 
 the same binding site in the canyon of mmoh . to test this possibility , 

we first investigated the binding competition between fd and the core 
 of mmob by fluorescence anisotropy titrations .
the mmob core was prepared 
 by truncating the n - terminal tail at residue 33 ( 2 - 33 ) , and 
 a cysteine mutation d36c was introduced in order to attach the fluorophore 
 iaedans .
the n - terminal truncated mmob ( designated mmob 2 - 33 ) 
 is still able to bind to mmoh but with lower affinity ( kd = 2.67 m , figure s3 ; kd = 0.55 m for the full - length 
 mmob ) . to characterize the binding competition
, 
 fd was titrated into a mixture of 1 m mmoh and 1 m iadeans - labeled 
 mmob 2 - 33 .
the fluorescence anisotropy decreased steadily as 
 increasing amounts of fd were added ( figure 5a ) , indicating displacement of n - terminal truncated mmob by fd from 
 mmoh ( scheme 1a ) .
the titration curve could 
 be simulated by assuming that the n - terminal truncated mmob and fd 
 compete for the same binding sites on mmoh ( mmohsite , one 
 on each side of mmoh ) , and an apparent kd value of 2 m was obtained for the mmoh 
fd complex 
 on the basis of the simulations ( figure 5a ) . experimental 
 evidence for mmor and mmob binding competition .
( a , b ) 
 titrating fd into 1 m mmoh and 1 m iadeans - labeled n - terminal 
 truncated ( 233 ) mmob ( a ) or full - length mmob ( b ) .
the 
 titration curves are simulated assuming kd = 0.9 , 2 , and 6 m for the mmoh  fd complex ; kd = 2 and 6 m best fit the experimental 
 data in ( a ) and ( b ) , respectively .
edc was used as the cross - linking reagent , and 0 , 1 , 2 , 4 , 
 and 6 equiv mmob relative to mmor were added to a mixture of 10 m 
 mmoh and 20 m mmor .
the cross - linking reaction mixtures were 
 resolved by sds - page , and bands corresponding to the mmor-subunit 
 cross - linking product are shown . in the binding competition 
 between fd and full - length mmob ( b ) , fd may displace the core of mmob 
 from the canyon , but the n - terminal tail may still bind to mmoh .
mmoh complex when the titration experiment 
 was repeated with the full - length mmob labeled with iaedans ( figure 5b ) , indicating that fd is less effective in displacing 
 full - length mmob .
this result is an expected consequence of the additional 
 binding site of the n - terminal tail of mmob on the surface of helices 
 h and 4 of the mmoh -subunit , a 
 binding region not shared with fd .
it is possible 
 that the n - terminal tail serves as an anchor attached to the surface 
 of mmoh , such that , when fd displaces the core of mmob from the canyon , 
 mmob does not completely dissociate from mmoh ( scheme 1b ) .
a simple competitive binding 
 model would therefore no longer apply to the component interactions 
 in this case .
similar results were obtained by using the full - length 
 mmor as the titrant . an apparent kd value 
 of 8 m for the mmoh  mmor complex is required to fit 
 the titration curve using a competitive binding model ( figure s4 ) , considerably larger than the 0.9 
 m kd value previously determined for the mmoh  mmor complex in the absence 
 of mmob .
these results clearly demonstrate that both fd and 
 the full - length 
 mmor are able to displace mmob , in particular the core of mmob , from 
 mmoh .
this property 
 was demonstrated by studying the effect of mmob on mmor cross - linking 
 to mmoh .
as shown in figure 5c , mmor forms 
 cross - links to the mmoh -subunit in the presence of edc , as 
 demonstrated previously ; the yield of 
 this cross - link decreases in a dose - dependent manner as the mmob concentration 
 increases from 0 to 6 equiv relative to the mmor concentration , indicating 
 that mmob blocks mmor binding , and thus its cross - linking , to mmoh . 
 a similar finding
the determination of 
 component protein binding sites on the hydroxylase and their binding 
 competition form a basis for understanding the role of the regulatory 
 protein in electron transfer reactions of smmo .
previously , mmob was 
 proposed to facilitate electron transfer from mmor to mmoh , and this 
 effect was ascribed to formation of a hypothetical mmoh2mmob2mmor 
 ternary complex . it was hypothesized 
 that the increased electron transfer exhibited by this ternary complex 
 is due to a conformational change of mmoh induced by mmor .
considering 
 the substoichiometric concentration of mmor found in vivo and used 
 during activity assays , it was further proposed 
 that such conformational changes are retained throughout the catalytic 
 cycle even after mmor dissociates from the ternary complex . considering our current finding that the core 
 of mmob and the fd of mmor compete for binding to the canyon of mmoh , 
 these proposals are deemed unlikely . because mmob can inhibit mmor 
 binding to the canyon
, mmob would inhibit electron transfer as well . to test experimentally whether mmob increases the electron transfer 
 rate , and whether pre - equilibrating mmor with other protein components 
 would result in such an increase , we designed two sets of single - turnover , 
 single - mixing stopped - flow experiments . in the first set ,
the electron 
 transfer reactions were initiated by mixing 2 equiv of chemically 
 reduced fd to ( i ) 1 equiv of oxidized mmoh alone , ( ii ) 1 equiv of 
 oxidized mmoh equilibrated with 2 equiv of mmob , and ( iii ) 1 equiv 
 of oxidized mmoh equilibrated with 2 equiv of mmob as well as 0.5 
 equiv of mmor ( figure 6a ) .
the reactions were 
 monitored at 470 nm , which increases in intensity as electrons are 
 transferred to the mmoh diiron sites from the reduced [ fe2s2 ] clusters .
the previous proposal would predict that 
 case ( iii ) , where all three components were pre - mixed and equilibrated , 
 would exhibit the fastest electron transfer rate . the actual results 
 ( figure 6b ) , however , showed that reaction 
 ( i ) displays the fastest electron transfer rate . including mmob in 
 reactions ( ii ) and ( iii )
significantly retarded the electron transfer 
 reaction , even when mmor was added and pre - equilibrated with other 
 components .
these 
 experiments were repeated using chemically reduced full - length mmor 
 as the reductant , and the same trend was observed ( figure s5 ) .
mmob inhibits electron transfer , using chemically reduced 
 fd as 
 the electron source .
the electron transfer reaction 
 was initiated by mixing 2 equiv of nadh with ( i ) 1 equiv of oxidized 
 mmoh and 2 equiv of mmor and ( ii ) 1 equiv of oxidized mmoh , 2 equiv 
 of mmob , and 2 equiv of mmor ( figure 7a ) .
the 
 reactions were monitored at 470 nm , where the absorption first decreased 
 due to the reduction of the fad cofactor by nadh and the subsequent 
 intra - mmor electron transfer to the [ fe2s2 ] 
 cluster .
the absorption at this wavelength 
 then increased , as electrons were transferred from these cofactors 
 to the diiron centers in mmoh .
the previous proposal would predict 
 that reaction ( ii ) , which had all three protein components pre - equilibrated , 
 would display the faster electron transfer rate , but in fact it is 
 slower ( figure 7b ) , again demonstrating the 
 inhibitory effect of mmob on electron transfer ( table 1 ) .
electron transfer reactions were 
 initiated by mixing the reagent in syringe 1 with protein(s ) in syringe 
 2 in single - mixing stopped - flow experiments ( setups also shown in 
 figures 6a and 7a ) .
the inhibitory effect of mmob on electron transfer 
 is in full agreement with our finding that the core of mmob shares 
 a binding site on mmoh with the fd of mmor such that mmob inhibits 
 mmor binding . although electron transfer is retarded , it is still 
 much faster than the rate of substrate turnover ( kcat = 0.1 s at 15 c using propylene 
 as substrate ) .
the advantage of such competitive binding is that it 
 provides a mechanism for modulating electron transfer during catalysis . 
 by fine - tuning the affinity of mmor and mmob for the canyon region 
 of mmoh , the binding of mmor can be inhibited during dioxygen activation , 
 preventing undesired electron transfer that could quench activated 
 dioxygen intermediate species .
such quenching is suggested by oxidase 
 activity displayed by mmoh and mmor in the absence of mmob .
the modulation of electron transfer exhibited 
 by the mmob regulatory protein is most likely a feature common to 
 other bmm enzymes .
these enzymes share conserved protein sequences 
 as well as structures , and , like smmo , their regulatory 
 proteins also bind to an analogous canyon region of the hydroxylase , 
 as illustrated in the x - ray structures of the protein complexes in 
 ph and t4mo , blocking the shortest pathway to the hydroxylase diiron center from 
 its surface .
the reductase may therefore need to compete with the 
 regulatory protein for binding in order to deliver electrons to the 
 diiron centers in these enzymes as well . in disagreement with this 
 hypothesis , previous studies of ph and tomo
these electron transfer rates , however , were determined 
 in steady - state assays in the absence of substrates , by measuring 
 nadh consumption . under these conditions ,
the nadh consumption rate 
 depends on the oxidase activity of the hydroxylase , and the accelerated 
 nadh consumption in the presence of the regulatory component is most 
 likely due to increased oxidase activity and not an indication of 
 accelerated electron transfer .
using 
 hdx - ms , the binding site for the ferredoxin domain of mmor 
 was determined to be in the canyon of mmoh , the same region where 
 the core of the mmob regulatory protein binds .
this finding is consistent 
 with previous chemical cross - linking results and the current computational 
 docking study , as well as a series of binding competition assays . 

mmob inhibits mmor binding to the canyon as well as the electron transfer 
 that leads to reduction of the hydroxylase .
the previous proposals 
 that mmob increases the electron transfer rate when all three components 
 are pre - equilibrated has been tested experimentally and proved to 
 be invalid .
regulatory proteins of other bmm enzymes may similarly 
 share binding sites with their reductases and inhibit electron transfer .
such binding competition would provide a control mechanism for 
 electron transfer in bmm enzymes . to initiate the catalytic cycle
, 
 the reductase displaces the regulatory protein from the canyon of 
 the hydroxylase , reducing the diiron center from diiron(iii ) to diiron(ii ) ; 
 the regulatory protein then re - binds to the canyon and displaces the 
 reductase , initiating o2 activation and substrate oxidation . 

the binding affinities of the component proteins may be fine - tuned 
 so that the reductase is unable to displace the regulatory protein 
 at this step , preventing quenching of activated oxygen intermediates 
 p * , hperoxo , and q. at the 
 end of the catalytic cycle , the diiron center returns to the diiron(iii ) 
 state , and the reductase can bind again to the canyon , priming the 
 enzyme for the next cycle of catalysis .
 the 
 hydroxylation or epoxidation of hydrocarbons by bacterial multicomponent 
 monooxygenases ( bmms ) requires the interplay of three or four protein 
 components . 
 how component protein interactions control catalysis , 
 however , is not well understood . 
 in particular , the binding sites 
 of the reductase components on the surface of their cognate hydroxylases 
 and the role(s ) that the regulatory proteins play during intermolecular 
 electron transfer leading to the hydroxylase reduction have been enigmatic . 
 here 
 we determine the reductase binding site on the hydroxylase of 
 a bmm enzyme , soluble methane monooxygenase ( smmo ) from methylococcus 
 capsulatus ( bath ) . 
 we present evidence that the ferredoxin 
 domain of the reductase binds to the canyon region of the hydroxylase , 
 previously determined to be the regulatory protein binding site as 
 well . 
 the latter thus inhibits reductase binding to the hydroxylase 
 and , consequently , intermolecular electron transfer from the reductase 
 to the hydroxylase diiron active site . 
 the binding competition between 
 the regulatory protein and the reductase may serve as a control mechanism 
 for regulating electron transfer , and other bmm enzymes are likely 
 to adopt the same mechanism . 
Introduction
Experimental Section
Results and Discussion
Conclusions