cytochrome b5 ( cyt b5 ) reacts with myoglobin ( mb ) 
 as part of a physiological repair system in which autooxidized and 
 inactive femb is reduced to its active femb o2-storage form by electron transfer ( et ) from fecyt b5 . the 
 [ mb : cyt b5 ] complex has served as a paradigm 
 for interprotein et between dynamically docked partners whose binding 
 and reactivity are decoupled ; many conformations of the complex contribute 
 to binding but few to et . in recent years , 
 we have demonstrated that binding and reactivity can be enhanced and 
 coupled in the [ mb : cyt b5 ] complex through 
 mutations on the mbwt surface and/or heme neutralization . these generate 
 a suite of positively charged mb s whose binding to the negatively 
 charged b5 increases with the charge product 
 of the proteins ( qmbqcyt  b5 ) ; et between 
 the mb s and cyt b5 was probed 
 through replacement of the heme group in mb with a porphyrin containing 
 a closed - shell metal ion ( zn ) , with the focus being photooxidation 
 of the photoexcited triplet state , znmb . most of this 
 work studied znmb by fecyt b5 on the time scale of milliseconds  seconds . however , 
 photoexcitation of znmb directly populates the first - excited singlet 
 state , s1 , and we discovered interprotein photooxidative 
 singlet et within a redesigned [ znmb : cyt b5 ] complex at rate constants that approach those occurring within 
 the photosynthetic reaction centers , ket = 2.1  10 s. in the rich history of photoinitiated interprotein 
 et where one partner is a metal - substituted hemoprotein ( m = h2 as well as mg , zn ) , the exclusive focus has been on oxidative 
 et quenching of the photoexcited protein partner , despite the fact that photoreduction of m - porphyrins is energetically 
 favorable , and that there have been reports 
 of reductive and oxidative quenching of photoexcited zn - substituted 
 proteins by small molecules . we report here that 
 photoinitiated electron flow involving a metal - substituted hemoprotein 
 and its physiological partner protein can indeed be the [ mb : cyt b5 ] complex stabilized by 
 three d / e  k mutations on mb ( mmb(d44k / d60k / e85k ) , denoted 
 mmb ; m = mg or zn ) exhibits both oxidative and reductive 
 et quenching of both the singlet and triplet photoexcited 
 mmb states , the direction of flow being determined by the oxidation 
 state of the cyt b5 partner . we find that 
 the first - excited singlet state , s1 , of the mb - incorporated 
 metalloporphyrin , denoted mmb , undergoes nanosecond - time 
 scale reductive et quenching in which an electron is transferred tommb from fecyt b5 , as well as nanosecond - time scale oxidative et quenching 
 in which an electron is transferred frommmb to fecyt b5 . surprisingly , 
 the rate constants for the two intracomplex reactions are the same 
 within error , with average rate constants , kf  10 s , a phenomenon 
 that is explainable on energetic grounds . the progress curves in both 
 cases are nonexponential and viscosity independent , implying the presence 
 of ensembles of structures that do not interconvert on the time scale 
 of the measurement . the triplet state of the mutant , mmb , created by intersystem crossing ( isc ) from mmb likewise 
 undergoes reductive et quenching by the fecyt b5 as well as oxidative et quenching by the fecyt b5 . again , surprisingly , 
 the rate constants for the two intracomplex reactions are the same 
 within error , kf  
 10 s , despite a large disparity in 
 driving forces . however , in this case the shape and viscosity dependence 
 of the progress curves indicate that et is gated by intracomplex conversion 
 among a conformational ensemble . the protocols for expression , reconstitution 
 with the desired metalloporphyrin , m ( m is mg - protoporphyrin ix = 
 mg or zn - deuteroporphyrin ix = zn ) , and purification of mb(d44k / d60k / e85k ) 
 have been outlined elsewhere , and are briefly described 
 in the expanded materials and methods section in supporting information ( si ) . the tryptic fragment of bovine 
 cyt b5 was isolated and purified as described 
 previously . aerobic cyt b5 prefers the ferric state because of its slight negative 
 redox potential ( 0.006 v vs nhe ) . however , for oxidative quenching experiments in which cyt b5 served as the oxidizing agent , it was further 
 treated with excess k3[fe(cn)6 ] and then washed thoroughly with working buffer ( 5 mm kpi , ph 6 ) . 
 for reductive quenching experiments in which cyt b5 served as the reducing agent , it was treated with excess 
 na2s2o4 and then washed thoroughly 
 with working buffer . the working buffer ( 5 mm kpi , ph 6.0 or 70% w / w glycerol 
 in 5 mm kpi , ph 6 ) was syringe - filtered and allowed to deoxygenate 
 in the glovebox for at least 24 h before the samples were made . protein 
 stock solutions were exchanged into the anaerobic working buffer using 
 corning spin - x uf concentrators immediately prior to the measurements . three types of sample were prepared : mmb by itself , cyt b5 by itself , and the complex , [ mmb : cyt b5 ] . the details concerning sample volumes and concentrations 
 for femtosecond- and nanosecond - transient absorption ( ta ) can be found 
 in si . the 120 fs pulses 
 were produced with a commercial ti : sapphire oscillator / amplifier ( tsunamic / spitfire , 
 spectra - physics ) , generating 1 w at 827 nm , operating at 1 
 khz . about 40% of this output was frequency - doubled and directed to 
 a two - stage opa producing pulses of 540 nm ( znmb samples ) or 598 nm 
 ( mgmb samples ) . five percent of the amplified pulse was sent up and 
 down a motorized delay track which provided the desired time resolution , 
 then was focused onto a sapphire disk to create a white - light continuum 
 probe with coverage from 430 to 850 nm . after passing through the 
 sample , the probe beam was dispersed onto a cmos array detector for 
 the collection of spectral data at multiple delay times following 
 photoexcitation of the sample . transient absorption 
 spectra were obtained by chopping the pump beam at 500 hz and subtracting 
 pump - on versus pump - off spectra . progress curves were generated 
 at multiple wavelengths from the ta spectra and fit using an exponential 
 ( for mmb decay ) or a stretched exponential ( for mmb reaction with cyt b5 ) ( see results and discussion ) . samples were excited with a nd : yag quanta - ray indi laser 
 ( spectra - physics ) tuned to 532 nm . the 
 output power was set to approximately 20 mw for the mgmb samples . triplet measurements were performed with an lks.60 laser flash photolysis 
 spectrometer ( applied photophysics ) fitted with a xenon lamp with 
 pulsing capabilities as the probe source . millisecond 
 collection mode uses an agilent infiniium 600 mhz digitizer with a 
 five - stage 1p28 photomultiplier tube as the detector . the triplet decay time 
 courses were monitored at 465 nm , the maxima for the triplet - ground 
 spectra difference for these samples . as decay traces span several orders of magnitude 
 in time , 50100 shots were averaged for each time - segment and 
 then merged into single files to obtain full kinetic progress curves 
 for analysis . the driving forces ( g ) for the charge separation reactions generated by the photoinitiated 
 oxidation or reduction of mgmb by ferric cyt b5 or ferrous cyt b5 , calculated 
 as described in si , are presented within 
 the et cycle of scheme 1 and in table 1 . the driving forces for charge recombination follow 
 from closing the thermodynamic cycle and are also given in scheme 1 and table 1 . both oxidative and reductive et quenching of mgmb by fecyt b5 ( right side of scheme 1 ) and fecyt b5 ( left side of scheme 1 ) , respectively , the photoinitiated oxidation 
 or reduction charge separation reactions of znmb by ferric 
 or ferrous cyt b5 are comparably energetically 
 favorable ( scheme s1 in si ) . the corresponding driving forces ( g ) for mgmb oxidation or reduction 
 charge separation reactions with ferric or ferrous cyt b5 were calculated analogously ; again , both oxidative and 
 reductive et quenching of mgmb by cyt b5 are highly favorable ( table 1 ; scheme s2 in si ) . mgmb forms 1:1 cyt b5 complexes with 
 dissociation constant , kd  10 
 m , as does znmb . figure 1 displays the absorbance - difference spectra in the 
 450600 nm region , collected subsequent to laser excitation 
 over the time scale of s1 decay for ( i ) mgmb by itself 
 ( top panel , figure 1 ) and for complexes with 
 ( ii ) fecyt b5 ( middle , figure 1 ) and ( iii ) fecyt b5 ( bottom , figure 1 ) ; the concentrations 
 of cyt b5 were chosen so that  90% 
 of the mmb was in complex . as can be seen , the singlet - ground absorbance - difference 
 spectra at t = 1 ps for the free mgmb and for the 
 two complexes are essentially the same ; the same is true for znmb 
 ( figure s1 in si ) . the traces of figure 1 show the mgmb difference spectrum collected 
 out to 5.2 ns ; the absorbance difference persisting after this time 
 is due to the triplet - ground difference generated by mgmb 
 that undergo isc to mgmb , which does not decay on the 
 ns singlet time scale . difference spectra for mgmb by itself ( top ) , 
 in complex with fecyt b5 ( middle ) , 
 and in complex with fecyt b5 ( bottom ) . as clearly seen in figure 1 , the mgmb excited state is strongly 
 quenched by complex formation with the cyt b5 partner protein in both its fe and fe oxidation states . analogous behavior is observed for znmb ( figure s1 in si ) , but mgmb decays approximately 5 times more slowly than znmb , 
 allowing for relatively clearer characterization of mgmb 
 et quenching , and consequently , we focus on this variant here . the 
 progress curves for the decay of mgmb , both free and in 
 the two complexes were assembled from the absorbance - difference spectra 
 slices at 465 nm and are shown in figure 2 . see figure s2 in si for progress curves 
 of free znmb and in complex with fecyt b5 and fecyt b5 . the free mmb ( m = mg , zn ) decays exponentially 
 ( eq 1),1where a0 is the singlet - ground absorbance difference , kd is the singlet decay constant , 
 and c is the absorbance difference associated with 
 the triplet state , which does not decay measurably on this time scale progress curves for singlet to ground 
 decay for mgmb ( gray ) , reductive quenching of mgmb in the presence of fecyt b5 ( blue ) , and oxidative quenching of mgmb in the 
 presence of fecyt b5 ( red ) . the mgmb trace is described by eq 1 , while the [ mgmb : fecyt b5 ] and [ mgmb : fecyt b5 ] traces are best described by eq 2 . for both the [ mmb : fecyt b5 ] and [ mmb : fecyt b5 ] complexes , the strong quenching of mmb by the cyt b5 ( figure 1 , figure s1 in si ) generates rapidly 
 decaying progress curves ( figure 2 , figure 
 s2 in si ) that are well described by augmenting 
 the intrinsic decay with a stretched exponential ( eq 2).2 this formulation describes an ensemble of complexes 
 that exhibit a distribution in quenching constants around an average 
 value , kf , the breadth of the distribution is reflected in the distribution 
 exponent , 0 < n  1 , with smaller values 
 for n corresponding to greater breadth of the distribution . the quenching constants for both mmb s obtained by globally fitting the progress curves 
 of figures 2 and s2 in si at multiple wavelengths to eq 2 are 
 presented in table 2 ; we note that the quenching 
 constants for znmb are less reliably obtained because 
 of the more rapid singlet decay . given the high driving forces for both oxidative and reductive et ( table 1 and scheme 1 ) we attribute the s1 quenching in both [ mgmb : cyt b5 ] complexes to intracomplex et : to photooxidation of the strongly reducing singlet excited 
 state of mgmb through mgmb  fecyt b5 et ( scheme 1 , right - side 
 of the photocycle ) with the rate constant of kf , ox ; to photoreduction of the s1 state by fecyt b5  mgmb et ( scheme 1 , left - side 
 of the photocycle ) with the rate constant of kf , red . likewise , the s1 quenching in znmb complexes 
 is attributed to intracomplex et due to photooxidation or photoreduction 
 with fecyt b5 or fecyt b5 , respectively ( si ) . as confirmation , analysis presented in si demonstrates that resonance energy transfer from the excited mgmb / znmb to fe or fecyt b5 can not be a significant component 
 of the quenching . due to 
 less cleanly resolved quenching in the znmb complexes , the distribution 
 exponent was fixed in eq 2 for [ znmb : fecyt b5 ] to the value obtained 
 from fits to the [ mgmb : fecyt b5 ] progress curves . efforts 
 to measure the rate constant for charge recombination of the charge 
 separated intermediates generated by et between mgmb / znmb and cyt b5 were not successful . such measurements were carried out in an earlier study of photooxidation 
 of a more tightly bound mmb variant , but that study showed that the charge recombination ( back 
 et ) reactions are faster than the charge separation ( or forward et ) 
 reactions , suppressing accumulation of the intermediate on the singlet 
 time scale as well as the triplet time scale as discussed below . in 
 the present study , this obstacle was compounded by the fact that in 
 the wavelength range monitored ( 450750 nm ) there is no singlet / ground 
 isosbestic point that could be used to optimally detect and monitor 
 the intermediate , and the absorbance - difference for the charge - separated 
 species is small relative to the singlet - ground absorbance difference 
 throughout the range . surprisingly , the average rate constants 
 for the photooxidation and photoreduction of mgmb , respectively 
 within the complexes with fecyt b5 and fecyt b5 , are 
 roughly equal , kf , ox  kf , red , despite the fact that 
 the driving forces for the two charge separation processes differ 
 by 0.4 ev ( table 1 ) . likewise , the average 
 photooxidation and photoreduction rate constants of znmb 
 are also similar ( table 2 ) . in short , the average 
 et quenching rate constant appears surprisingly insensitive to the 
 direction of the electron flow being essentially the same when mmb is oxidized by fecyt b5 or reduced by fecyt b5and kf is further insensitive to the identity of m ( table 2 ) . the fits of the et quenching to a stretched 
 exponential with n < 1 imply the presence of an 
 ensemble of complexes with a distribution of singlet et rate constants . brownian dynamics ( bd ) simulations , indeed predict the presence of 
 such an ensemble , with m  fe distances that range from 15 to 
 20  and a shortest reactivity distance of 15.3  for the 
 complexes between the mb mutant and cyt b5 . however , the observation of a distribution 
 of quenching constants , rather than an average over the rate constants 
 of the ensemble , further implies that the ensemble of bound complexes 
 do not undergo conformational interconversion on the et time scale . 
 to test this implication , et was measured for the [ mgmb : fecyt b5 ] and [ mgmb : fecyt b5 ] complexes in 70% w / w glycerol buffer , where 
 the viscosity is 20 greater than that of aqueous buffer . figure 3 shows that the singlet kinetic data 
 collected in glycerol and aqueous buffers overlay well for both oxidative 
 and reductive quenching , and et kinetic parameters for the two solvents 
 differ insignificantly , confirming the absence of any role for conformational 
 interconversion within the ensemble of structures during the nanosecond 
 duration of the singlet et charge separation process . progress curves for oxidative 
 ( red ) and reductive ( blue , offset by 0.3 arbitrary absorbance 
 units ) quenching of mgmb by fe and fecyt b5 , respectively in 70% gly 
 and aqueous buffer . we now show that , 
 when the et energetics for oxidation and reduction charge separation 
 processes ( table 1 ) are incorporated in the 
 marcus equation , they fully account for the unexpectedly similar rate 
 constants for the photooxidation and photoreduction of mmb within the complexes with fecyt b5 and fecyt b5 , respectively . marcus showed that an et rate constant can be written 
 as the product of two terms  the tunneling matrix element ( hda2 ) and the franck the 
 former is a joint function of the distance between the donor and acceptor 
 and the electron - transfer pathways ( dictated by the protein as the 
 intervening medium in this case ) , while the latter is a function of 
 the driving force ( g ) 
 for the electron transfer reaction , eqs 3,3a3bfc ( eq 3b ) is a  simple parabolic function of the driving force ( g ) for et with maximum at g =  ( where  is the reorganization energy ) . figure 4 plots fc as a function 
 of g for   
 1 ev ; previous reports have demonstrated 
 that the same  value is applicable to both oxidative and reductive 
 charge recombination reactions for the same photoexcitable species . fc term as a function of driving forces 
 ( g ) and  = 1.0 
 ev for et in the mgmb complexes with cyt b5 . oxidative quenching or et from mgmb to fecyt b5 is in red and reductive quenching 
 or et from fecyt b5 tomgmb is in blue . since g = 0.8 ev ( see table 1 ) for reductive quenching 
 due to singlet et and oxidative quenching due to triplet et , the fc terms for these two processes are offset by 0.1 in opposite 
 directions for clarity . while the [ mgmb : fecyt b5 ] and [ mgmb : fecyt b5 ] complexes are expected to exhibit a similar ensemble of structures 
 and electron - transfer pathways ( due to the same ranges of reactivity 
 distances and the same protein media ) and , therefore , comparable hda2 , the driving forces for the oxidative and reductive mgmb et charge separation processes differ by (g )  0.4 ev ( table 1 ) . however , figure 4 highlights the unexpected 
 fact that the two driving forces ( depicted by circles ) are symmetrically 
 placed around and near the maximum of the parabola at g =  , oxidation being in the  inverted  
 region , g > < . as a result , fc for the two processes is fortuitously the same . moreover , since 
 the two driving force values fall near the fc maximum , 
 slight adjustments of either the g s or  would likely result in the same 
 set of kinetic observations . thus , the apparently surprising similarities 
 in the kf for the oxidative 
 and reductive quenching of mgmb are understandable on 
 simple energetic grounds . s for singlet et charge separation processes 
 in the [ znmb : fecyt b5 ] and [ znmb : fecyt b5 ] complexes are symmetrically related to  ( figure 
 s4 in si ) , again explaining why the oxidative 
 and reductive singlet et quenching reactions have similar kf s ( table 2 ) . additionally , the respective oxidative and reductive et charge 
 separation processes for the [ znmb : cyt b5 ] complexes have driving forces that are within (g )  0.1 ev ( scheme s1 in si ) of those for the [ mgmb : cyt b5 ] complexes ( scheme 1 ; table 1 ) , accounting for the m - independence 
 of et rate constants . examination of the parameter characterizing 
 the breadth of the distribution in structures ( n , 
 eq 2 ) does , however , reveal a slight oversimplification 
 in the above comments about hda2 . for both m = mg and zn , the 
 distribution parameter for oxidative singlet et quenching ( n  0.75 ) appears to be meaningfully different than 
 that for reductive singlet et quenching ( n  
 0.50 ) suggesting a broadening of the distribution of rate constants 
 for reductive quenching in the complex , [ mgmb : fecyt b5 ] , relative to oxidative quenching 
 in the complex , [ mgmb : fecyt b5 ] ( table 2 ) . this dissimilarity 
 can be attributed to a subtle variance in the ensemble of binding 
 geometries resulting from the difference by a single charge in the 
 two oxidation states , ferrous and ferric , of the highly negative cyt b5 partner in the two types of complexes . conformations of the [ mgmb : cyt b5 ] complexes that do not react 
 via singlet et ( or relax to ground ) instead undergo isc to the [ mgmb : cyt b5 ] state , which can 
 react via photooxidation or photoreduction of mgmb by 
 the fecyt b5 and fecyt b5 , respectively , on the much longer 
 ( ms ) triplet - state time scale . figure 5 presents 
 progress curves for triplet decay monitored at 465 nm , the maximum 
 triplet - ground difference absorption wavelength , for samples of the 
 [ mgmb : fecyt b5 ] complex and 
 the [ mgmb : fecyt b5 ] complex 
 in aqueous and 70% w / w triplet progress curves monitored at 465 nm : 
 mgmb by itself in the absence of cyt b5 quencher in gray ; in the presence of fecyt b5 in blue ; in the presence of fecyt b5 in red . triple et slows down with 
 increased viscosity as noted by the rightward shift ( toward kd ) of the progress curves in 70% gly 
 relative to the ones in aqueous buffer . in the absence of cyt b5 , mgmb decays to the ground state with first - order rate constant , kd = 400 s independent of solvent ( figure 5 ) . as previously reported , the [ mb : cyt b5 ] complex is in the slow - exchange regime on the triplet et time scale 
 where et is significantly faster than dissociation of the complex , ket  koff . as a result , triplet 
 decay traces for the complex are biphasic ( figure 5 ) and can be fit to a double exponential ( eqs 4 ) : the rapidly decaying majority phase corresponds to the 
 [ mgmb : cyt b5 ] complex ( fraction r ) , which decays with rate constant , kobs , f = kd+kf where kf is 
 the intracomplex triplet et rate constant ; the minority phase represents 
 free mgmb , which undergoes slower bimolecular quenching 
 ( k2 ) with pseudo - first - order 
 rate constant kobs , s = kd+k2*[cyt b5].4awhere the kinetic parameters 
 resulting from fits to eqs 4 are listed in 
 table 3 . monitoring the triplet / ground isosbestic 
 points yielded no quantifiable charge - separated intermediate signals 
 for either the oxidative or reductive quenching charge separation 
 triplet et reactions , supporting the expectation that the accumulations 
 of the respective charge - separated species are even smaller on the 
 triplet time scale relative to the singlet time scale due to the relative 
 lifetimes of the charge separation and charge recombination processes . the rate constants for intracomplex triplet 
 et , kf s , are essentially 
 the same for the oxidative and reductive processes in the same type 
 of solvent ( table 3 ) , but in this case the 
 identity is not in agreement with predictions of the marcus theory 
 based on the energetics for triplet et quenching . as with singlet 
 et , triplet et is energetically favorable for both oxidative quenching 
 ( g = 0.8 ev ) and reductive 
 quenching ( g = 0.4 ev ) 
 of the mgmb with the difference between these two driving 
 forces also being 0.4 ev ( table 1 ) . 
 at the same time , the relative driving forces ( g ) are not symmetrically positioned around the 
 estimated  ( figure 4 ) , and the fc , and ket is predicted 
 to differ by more than an order of magnitude for the two charge separation 
 processes : ket , ox > ket , red . the 
 similarity of the two kf s 
 instead are explained by comparing the progress curves for the complexes 
 in aqueous (   1 ) and 70% w / w glycerol buffers (  
  20 ) . as expected , the diffusion - limited second - order process 
 slows in 70% w / w gly relative to water for both the photooxidative 
 and photoreductive reactions with k2 decreasing by a factor of  20 as the viscosity increases 
 by about this factor ( table 3 ) . unexpectedly , 
 the rate constants for both oxidative and reductive intracomplex et 
 decrease by roughly the same factor ( table 3 ) . this equality and viscosity - dependence of the rate constants for 
 intracomplex oxidative and reductive triplet et , kf , ox  kf , red , demonstrates that the measured intracomplex rate 
 constant for charge separation reflects not the et process itself 
 but rather the rate constant for interconversion among conformations 
 of the bound complex ; conformational interconversion on the s ms 
 time scale serves as a  gate  to the intracomplex charge 
 separation triplet et . we have 
 shown that photoinitiated electron flow within the [ mmb : cyt b5 ] complex ( m = mg or zn ) that is stabilized 
 by three d / e  k mutations of mb can indeed be  symmetrized  ; 
 the complex exhibits both oxidative and reductive 
 et quenching of both the singlet and triplet photoexcited 
 mmb states , with the direction of electron flow being determined by 
 the oxidation state of the cyt b5 partner , 
 fecyt b5 or fecyt b5 . despite a large disparity in 
 driving force in favor of photooxidation relative to photoreduction 
 ( (g )  0.4 
 ev , m = mg ;  0.2 ev , m = zn ) , the ultrafast intracomplex reductive 
 and oxidative et quenchings of mmb surprisingly have the 
 same average et rate constants of kf  10 s. equally surprising , 
 the intracomplex reductive and oxidative et quenchings of mmb are again the same within error , kf  10 s. the equality of the rate constants for photooxidation and photoreduction 
 of mmb is explainable on energetic grounds ; the driving 
 forces for the two reactions have values symmetrically displaced above 
 and below that of the reorganization energy ,  , and thus equal 
 franck  condon factors ( fc ) within the marcus 
 equation . the progress curves for both directions of electron flow 
 during et quenching of mmb are nonexponential and viscosity 
 independent , implying the presence of an ensemble of structures that 
 do not interconvert on the time scale of the measurement . the 
 rate constants for photooxidation and photoreduction of mmb are again equal despite a large difference in driving force , 
 but in this case the driving force difference predicts a large difference 
 in fc and thus in rate constant . this behavior is 
 explained by the findings that the progress curves are exponential 
 and the rate constants decrease inversely with viscosity , which indicate 
 that the quenching constants are not et rate constants at all , but 
 instead that the reactions are gated by intracomplex conformational 
 interconversion . the control of et on the time scale of triplet - state 
 quenching by conformational interconversion underscores the important 
 point that protein motions can control even intracomplex et reactions 
 when they occur on the s  ms time scale , suggesting that 
 reconsideration of numerous such earlier studies might be productive , 
 whereas the independence of the singlet - state et from such motions 
 indicates that they are not likely to affect the relatively few et 
 reactions on the ns time scale .
we report here that photoinitiated 
 electron flow involving a metal - substituted ( m = mg , zn ) myoglobin 
 ( mb ) and its physiological partner protein , cytochrome b5 ( cyt b5 ) can be 
 symmetrized  : 
 the [ mb : cyt b5 ] complex stabilized by 
 three d / e  k mutations on mb ( d44k / d60k / e85k , denoted mmb ) 
 exhibits both oxidative and reductive et quenching of both the singlet 
 and triplet photoexcited mmb states , the direction of flow being determined 
 by the oxidation state of the cyt b5 partner . 
 the first - excited singlet state of mmb ( 1mmb ) undergoes 
 ns - time scale reductive et quenching by fe2+cyt b5 as well as ns - time scale oxidative et quenching 
 by fe3+cyt b5 , both processes 
 involving an ensemble of structures that do not interconvert on this 
 time scale . despite a large disparity in driving force favoring photooxidation 
 of 1mmb relative to photoreduction ( (g0 ) 
 0.4 ev , m = mg ;  0.2 ev , 
 m = zn ) , for each m the average rate constants for the two reactions 
 are the same within error , 1kf > 108 s1 . 
 this surprising observation 
 is explained by considering the driving - force dependence of the franck 
 condon 
 factor in the marcus equation . 
 the triplet state of the myoglobin 
 ( 3mmb ) created by intersystem crossing from 1mmb likewise undergoes reductive et quenching by fe2+cyt b5 as well as oxidative et quenching by fe3+cyt b5 . as with singlet et , 
 the 
 rate constants for oxidative et quenching and reductive et quenching 
 on the triplet time scale are the same within error , 3kf  105 s1 , but here the equivalence is attributable to gating by intracomplex 
 conversion among a conformational ensemble .