one of the main roles 
 of oxidative phosphorylation in mitochondria 
 and bacteria is maintaining a proton - motive force ( pmf ) across the 
 inner or plasma membrane , thereby providing most of the energy for 
 the synthesis of adenosine triphosphate ( atp ) in aerobic environments . the pmf is formed by transferring or pumping protons across the lipid 
 membrane , generating a transmembrane electrochemical potential and 
 transmembrane ph difference ( ph ) . during aerobic respiration , 
 electron - transport chains of oxidative phosphorylation terminate with 
 heme - copper oxygen reductases ( hcos ) , in which a heme - copper binuclear 
 center ( bnc ) binds and reduces dioxygen to water . complex 
 iv and a bacterial homologue , cytochrome bo3 from escherichia coli , belong to the a1 subgroup 
 of type - a hcos , which typically pump one proton per electron , therefore 
 four protons per dioxygen molecule . the 
 reduction of dioxygen is further coupled to the uptake / release of 
 four charges ( protons and/or electrons ) , adding up to a total of eight 
 charges transported across the membrane for each dioxygen that is 
 reduced . type - a hcos possess two conserved proton channels , known 
 as the k- and d - channel , both leading to the bnc . the k - channel facilitates the 
 transportation of  chemical protons  that react with 
 dioxygen to form water ; the d - channel facilitates both the uptake 
 of  chemical protons  and the transportation of all 
 the pumped protons . both channels take up protons from the same side of the membrane : 
 the matrix side in mitochondria or the cytoplasmic side in bacteria . despite various high - resolution structures and extensive 
 biochemical and biophysical research , a distinct understanding of the molecular mechanism by which oxygen 
 reduction is coupled to proton pumping is still elusive , while the 
 manner by which turnover is controlled by the pmf remains relatively 
 unexplored . hcos , including cytochrome bo3 , have been observed to reduce activity in the presence of high pmf . pumping stoichiometry ( decrease 
 in the proton - to - electron ratio ) has been observed in many hco studies and also for other transport systems . the implications of these alterations in stoichiometry 
 have been previously discussed for hcos and for pumping atpases and 
 transporters in healthy and disease states . slips in substrate transport have been proposed to act as a  safety 
 valve  through which excess driving force can be dissipated and it is proposed to be a mechanism by which 
 hcos can optimize the condition for atp synthesis . maintaining such balance is important since high pmf and 
 a highly reduced electron - transport chain ( high ratios in ubiquinol / ubiquinone 
 and nadh / nad ) have been linked to increased production 
 of reactive oxygen species ( ros ) in mitochondria , which has been implicated in aging , cancer , and other diseases . proton uptake / release 
 activity of reconstituted cytochrome bo3 is controlled by reducing ubiquinone electrochemically ( a ) . the 
 change in intravesicular ph is determined via an encapsulated ph - sensitive 
 fluorescent dye , 8-hydroxypyrene-1,3,6-trisulfonic acid ( hpts ) ( a ) . a color - inverted fluorescent image ( b ) and the fitting of the fluorescence 
 of the circled proteoliposome by a gaussian function ( c ) ( the grid 
 is raw data , and the colored surface is the fitting result ) are shown . the sample image in ( b ) shows a small area of the whole image which 
 typically contains hundreds of ( proteo)liposomes . current understanding of the pumping mechanism of hcos primarily 
 relies on ensemble studies averaging the behavior of multiple pumps . while providing a great wealth of information , these studies average 
 out rare events during continuous turnover , masking the dynamic heterogeneity 
 of hcos and effectively preventing detection of leak states . single - molecule 
 studies on the other hand offer the exquisite ability to directly 
 observe the temporal fluctuations of individual enzymes and their 
 role in regulation of their activities . single - enzyme 
 studies were pioneered for ion channel current recordings and , more 
 recently , single - enzyme studies on transporters have visualized the 
 structural dynamics of transmembrane helices . here , we have developed a novel platform that 
 can directly observe the proton transfer of single cytochrome bo3 enzymes for prolonged periods ( up to 100 
 s ; figure 1a ) . our 
 studies reveal that cytochrome bo3 , a 
 representative hco enters a rare , long - lifetime leak state  
 during which protons rapidly leak or are transported back along the 
 transmembrane electrochemical potential and ph . our findings 
 support a view in which one of the underlying causes of the previously 
 observed reductions in the h / e stoichiometry 
 is a leak state , which provides new insights into the molecular mechanism 
 of proton pumping in hcos and its regulation by excessive ph . the novel method described in this study also adds to the ever expanding 
 library of techniques to study single molecules on surfaces by combining 
 optical and electrochemical methods . this study was carried out on cytochrome bo3 , an ubiquinol oxidase from e. coli . a key 
 difference between bo3 and cytochrome c oxidases such as complex iv is their electron donor , which 
 are ubiquinol and cytochrome c , respectively . this 
 allowed us to design a combined electrochemical - microscopic experimental 
 setup for single - enzyme analysis as schematically shown in figure 1 . in this setup , 
 which is described in detail in the materials and 
 methods , proteoliposomes containing cytochrome bo3 are sparsely adsorbed on a transparent ultrasmooth thin 
 gold electrode within an electrochemical cell . the gold is modified 
 with a self - assembled monolayer of 6-mercapto - hexanol , which renders 
 it hydrophilic and allows immobilization of the proteoliposomes , that 
 retain the ph sensitive fluorophore . to approach single - enzyme conditions , the proteoliposomes are made using a very low protein - to - lipid ratio . because of the low protein - to - lipid ratio , most of the liposomes in 
 the experiments do not contain a protein and we will adopt the term 
  ( proteo)liposomes  in the remainder of this text to 
 reflect this . image analyses procedures are used to exclude ( proteo)liposomes 
 that are close together by picking circular and separated bright spots 
 with a typical size that is consistent with single vesicles ( see materials and methods ) . nonetheless , this analysis 
 will not be able to detect if two ( proteo)liposomes are less than 
 200 nm apart . as described in the supporting information , simulations based on assumption that vesicles randomly adsorb on 
 the surface suggest that this will occur in only 0.3  0.2% of 
 fluorescent spots . because most ( proteo)liposomes do not contain an 
 enzyme , the probability that two proteoliposomes with an enzyme coincide 
 in a single fluorescent spot is much lower . the ( proteo)liposomes 
 also contain ubiquinone that is electrochemically 
 reduced to ubiquinol , thereby activating the oxygen reducing activity 
 of cytochrome bo3 . the ( proteo)liposomes 
 are loaded with a ph - sensitive fluorescent dye ( hydroxypyrene-1 , 3 , 
 6-trisulfonic acid trisodium salt ( hpts ) ) and the electrochemical 
 cell is placed in an epi - fluorescent microscope that monitors the 
 fluorescence which , with the use of separately determined calibration 
 data and the measured vesicle sizes , is converted to intravesicular 
 ph and the number of transported protons in individual ( proteo)liposomes 
 ( see supporting information for the calibration 
 procedure ) . example traces are 
 given for proteoliposomes where protons are pumped throughout the 
 application of the electrochemical potential ( a , b ) , proton leakage 
 ( c , d ) , proton uptake / release cessation without leakage ( e ) . an example 
 trace of a liposome without cytochrome bo3 is also shown ( f ) . red lines in the example traces ( a  f ) 
 are fits to the data ( gray ) as explained in the text . proton uptake / release 
 activity was initiated by applying an electrochemical potential between 
 time points indicated by the vertical dashed lines . figure 2 shows a 
 collection of typical ( proteo)liposome time traces , where the intravesicular 
 ph changes ( see figure s1 ) are used to 
 calculate the cumulative number of transported protons . because of 
 the low protein - to - lipid ratio used , the majority of the observed 
 ( proteo)liposomes were plain liposomes and did not portray any change 
 in ph upon reducing the ubiquinone pool electrochemically ( figure 2f ) . during the time 
 window the ubiquinone pool was electrochemically reduced ( dashed lines 
 in figure 2 ) , about 
 15% of the observed ( proteo)liposomes displayed significant ph changes 
 ( 0.1 < ph < 1 and in rare cases up to about 2 units ) . 
 indeed , the average number of cytochrome bo3 complexes per ( proteo)liposome is calculated to be 0.16 using the 
 average protein - to - lipid ratio and the average ( proteo)liposome size 
 ( see materials and methods for details ) . assuming 
 poissonian dilution of proteins / liposome we would then expect 15% 
 of liposomes to contain one active protein and 1% of liposomes 
 to have more than one protein . this calculation is concurrent with 
 our hypothesis that most ( proteo)liposomes displaying ph changes are 
 due to proton uptake / release by single enzymes , which however could 
 not be confirmed via direct experimental measurement . deviations from 
 the calculated average number or proteins / liposome are expected because 
 of the polydispersity in ( proteo)liposome diameter ( 71 nm with a full 
 width at half maximum ( fwhm ) of 45 nm , figure s8 ) and hence surface area . additional deviations at the level 
 of single particles could be present due to the heterogeneous distribution 
 of proteins and/or lipids . the ph values were observed to both increase 
 and decrease ( figure 2a and 2b , respectively ; hereafter these proteoliposomes 
 will be described 
 as  active  liposomes or vesicles ) , indicating cytochrome bo3 was present in both orientations in the proteoliposomes . about 76% of active vesicles show a ph increase consistent with a 
  right - side out  orientation . the single - enzyme traces 
 reveal a distribution in turnover rates ( figure 3 ) , while in more than 10% of cases , cytochrome bo3 was observed to abruptly halt proton uptake / release 
 ( figure 2e ) . such dynamic 
 heterogeneity and  stalling  of activity was observed 
 for enzymes in both orientations and is a feature typically observed 
 for ( single ) enzymes . importantly , however , 
 a significant fraction of our single enzyme traces ( 7.2  1.5% ) 
 also show abrupt turns in ph changes , rapidly destroying formed ph 
 even when the potential was still applied to reduce the ubiquinone 
 pool . such events were observed for cytochrome bo3 in both orientations , as shown in figure 2c and d. distribution of proton translocation rates 
 for ( top ) wild - type 
 cytochrome bo3 ( middle ) e286c cytochrome bo3 and ( bottom ) wild - type cytochrome bo3 in the presence of valinomycin . cytochrome bo3 was orientated both ways in the single - enzyme 
 proteoliposomes and relative frequencies are separated for the rates are determined from the slope of the analyzed 
 data for which examples are shown in figure 2 . the error bars represent the standard error 
 of the mean . in order to quantitatively analyze the heterogeneous activity of 
 the enzyme and resolve the distinct phases in the time traces ( e.g. , figure 2c  e ) without 
 human bias , an automatic trace analysis was coded and its results 
 are visualized by the red lines in figure 2 ( see materials and methods for details ) . to reproduce the findings , they were recorded in two 
 separate laboratories , first in copenhagen and then in leeds . two 
 different methods varying in experimental setup , ph - dependent fluorescent 
 probe ( hpts vs snarf-1 ) , and materials were used , which further support 
 their validity ( see the independent verification section in the supporting information ) . two different image 
 and data analysis procedures were also tested and compared , and both 
 methods confirmed the conclusions presented below . the data analysis 
 procedure resolves phases / events which are statistically distinctive 
 ( proton translocation , proton leakage and the stalling phase ) within 
 the time - traces and yields measurements such as the average proton 
 translocation rate for each event , the duration of the event , and 
 the ph at the onset of each event . cyclic voltammograms 
 ( cvs ) of absorbed proteoliposomes of wt and 
 e286c cytochrome bo3 . electrode preparation 
 and modification are identical to the single - enzyme experiments except 
 that 150 nm gold was used , proteoliposomes were reconstituted with 
 a higher protein - to - lipid ratio ( 1% w / w ) and full layer of proteoliposomes 
 were immobilized on the electrodes by incubating the electrode with 
 0.5 mg / ml proteoliposomes for 30 min . cvs ( 2 mv / s ) were measured under 
 ambient , aerobic conditions at 20 c . the onset of ubiquinol-10 
 reduction in the proteoliposomes starts at about 0.05 v vs 
 she . a cv of the electrode surface prior to immobilization 
 of proteoliposomes ( blank ) is also depicted . the proton translocation rates correspond to an ensemble 
 average 
 of 73  2.2 protons s ( figure 3 ) , which is less than the published activity 
 of 520 electrons s in reference and 850 electrons s in reference . we ascribe this difference to various reasons : 
 ( a ) only about half of the maximum activity of cytochrome bo3 is reached at the electrochemical potential 
 used to activate cytochrome bo3 in the 
 single - enzyme experiments ( 0.2 v vs standard hydrogen electrode 
 ( she ) , figure 4 ) and 
 ( b ) vmax condition can not be reached using 
 ubiquinol-10 as the electron donor . ubiquinol-10 is nearly identical 
 to the native electron donor in e. coli ( ubiquinol-8 ) . 
 however , previously reported values were measured using substrate 
 analogues ( decyl - ubiquinol or ubiquinol-1 ) under saturation conditions , while here ubiquinol-10 
 was added to the ( proteo)liposomes under nonsaturating conditions 
 ( 1% w / w ubiquinol-10/lipid ) . saturation of ubiquinol-10 in the membrane 
 is not possible due a disrupting effect of high ubiquinol-10 concentrations 
 and a previously reported substrate inhibition effect . analysis shows that 7.2  1.5% of 
 the proton - transporting 
 proteoliposomes abruptly switch to seemingly passive leaking within 
 the potential window . the reproducibility and statistical robustness 
 of our analysis is verified by 4 independent series of experiments 
 with cytochrome bo3 from two different 
 overexpressing strains , which show maximum and minimum leak probabilities 
 ranging from 2.9 to 9.5% . statistically , these leak events are inconsistent 
 with multiple enzymes per active vesicle , as it would require two 
 oppositely orientated cytochrome bo3 complexes 
 to behave in certain patterns , e.g. , one enzyme stopping and the other 
 starting at about the same time or the slower enzyme halting activity 
 first . to confirm that leak events are obtained from proteoliposomes 
 containing single enzymes , the experiments were repeated with an average 
 protein - to - lipid ratio 1 order of magnitude lower , where the stochastic 
 probability of having two oppositely oriented protein complexes is 
 2 orders of magnitude lower ( < 0.1% ) . as expected , the number of 
 ( proteo)liposomes displaying ph changes was significantly reduced 
 ( 2.5%  0.33% ) , but the abrupt switch was still observed for 
 3.2%  0.65% of the active vesicles ( 4 independent experiments 
 with leak state percentages between 1.9 and 4.5% ) . we propose that 
 these data further confirm that the leak activity is due to single 
 proteoliposomes with single enzymes . although it is known that 
 membrane pores can randomly form in the 
 presence of ph , pores are transient , submillisecond events . the observed lifetime of the leaking events ( > 10 s ) is thus inconsistent 
 with spontaneous pore formation in the lipid membrane of the vesicles . furthermore , the proton permeability of the reconstituted proteoliposomes 
 was independently measured by applying a ph to the reconstituted 
 proteoliposomes and empty liposomes under nonturnover conditions . 
 in both cases the recorded proton leaking 
 rate is significantly lower than the observed leak states of cytochrome bo3 ( figure s2 ) , confirming 
 that background proton permeability is negligible under our conditions . furthermore , the fact that we observe similar background proton permeability 
 for proteoliposomes reconstituted with cytochrome bo3 and empty liposomes verifies that leaking is not an 
 artifact of the reconstitution method . taken together the abrupt changes 
 in proton transport direction are most likely due to single enzyme - related 
 proton leakage into or out of the proteoliposomes . a simple 
 explanation for the observed leak events and stalling 
 is that they are due to irreversible inactivation or damage to cytochrome bo3 . because less than 10% of the proteoliposomes 
 enter the leak state and because the time - span of the experiments 
 is limited ( up to 100 s of active turnover ) it is not possible to 
 directly observe the rare recovery events . in addition , there is also 
 the possibility , albeit low , of proteoliposomes containing two enzymes 
 ( or two proteolipsomes are less than 200 nm apart ) and thus registering 
  false  recovery events . we therefore analyzed the relative 
 number of proteoliposomes in consecutive movies on the same electrode 
 ( figure s3 ) . the percentage of the proteoliposomes 
 containing cytochrome bo3 that actively 
 take up / release protons does not decrease when movies were taken sequentially . the movies were taken from different areas on the same electrode , 
 but lipid vesicles that were not being fluorescently imaged are still 
 subjected to the same electrical potential applied to the gold electrode 
 which would start the enzyme turnover . therefore , if cytochrome bo3 could not revert back to normal proton translocation 
 after entering the leak state or the stalling phase , the percentage 
 of active vesicles would have shown a continuous decline . the lack 
 of such downward trend indicates that both the leak state and the 
 stalling phase are reversible . it is generally accepted that 
 back - flow of protons during turnover 
 of hcos is prevented by a gate in the d - channel . we hypothesized that the long - lived leak events are caused by a 
 conformational change causing the gate to  open  allowing 
 protons to freely flow back . glutamic acid 286 ( e242 in bovine heart 
 complex iv ) is highly conserved in type a1 hcos and lies at the end 
 of the d - channel . it has been suggested 
 that e286 is an integral part of the gating mechanism and to test this hypothesis , the same measurement and analysis was 
 applied using an e286c mutant . in e286c cytochrome bo3 , proton translocation 
 through the d - channel is blocked and protons are prevented from being 
 pumped across the membrane . it is worth 
 noting that besides the pumped protons , redox reactions in cytochrome bo3 result in a further net translocation of 
 four  chemical protons  across the membrane : the reduction 
 of every dioxygen molecule draws four protons from the cytoplasmic 
 side and another four protons are released into the periplasm after 
 the oxidation of two ubiquinols . the e286c 
 mutant is catalytically active ( figure 3 and 4 ) and thus able to generate 
 a ph via the net translocation of  chemical protons  . we note here that ubiquinone is also protonated when electrochemically 
 reduced at the electrode . however , since electrons can not transfer 
 across the lipid bilayer , this process takes up protons from solution 
 only ( i.e. , from outside the vesicles ) and should therefore not influence 
 the intravesicular ph . the orientation of e286c cytochrome bo3 is similar to wild type ( wt ) : 66% of proteoliposomes 
 with e286c 
 cytochrome bo3 take up protons from the 
 inside of the lipid vesicle ( alkalinization ) , compared to 76% of wt . the oxygen - reducing activity of e286c cytochrome bo3 as measured by the electrochemical current ( figure 4 ) is about 1520% 
 of that of wild - type cytochrome bo3 , depending 
 on the potential at which the comparison is made . the lower proton - transporting 
 activity of e286c cytochrome ( 22% of that of wt , figure 3 ) is consistent with the lower 
 oxygen reducing activity , although higher than expected considering 
 the fact that e286c cytochrome bo3 can 
 only release / uptake one proton per electron rather than two for wild - type 
 cytochrome bo3 . this we attribute to the 
 data analysis and noise levels , as very low proton uptake / release 
 rates are hard to detect . the relative frequency ( in percentage ) of entering 
 the leak state 
 during turnover in relation to the leaking rate and these figures are 
 two - dimensional histograms generated in the same way as the conventional 
 one - dimensional histogram ( the color of the pixels is equivalent to 
 the height of the bars in histogram bar chart ) . the sum of the relative 
 frequency values in each heat map equals to the total relative frequency 
 of proteoliposomes entering the leak state in the corresponding experiment . the ph is the intravesicular ph minus the ph of the bulk solution 
 ( set constant at 7.4 ) . the single - enzyme experiments show that the relative frequency 
 by which e286c enters the leak state is significantly reduced to 0.6% 
 ( 0.5% and 1% in two independent data sets each containing eight and 
 20 movies , respectively ) , compared to 7.2% for wild - type cytochrome bo3 . besides the significantly reduced percentage 
 of leak events observed for e286c cytochrome bo3 , the leaking rates are also significantly lower for e286c 
 ( 25.5  4.1 h / s vs 102.3  11.2 h / s for the wild - type , figure 5 ) . we propose that these results further substantiate that 
 the leak events observed from wild - type cytochrome bo3 are caused by a leak state of the proton pump and not 
 by oppositely orientated enzymes or pore formation in the lipid membrane . although the results from the e286c mutant confirm that protons leak 
 through the protein , they do not unambiguously show that protons flow 
 via the d - pathway and more work is needed to identify the leak pathway . 
 because this mutant is less active than wt , the e286c mutation could 
 allosterically reduce the probability that a leak state is formed . indeed , on average , the lifetime of proton uptake / release in e286c 
 cytochrome is about 25% longer than wt ( figure s4 ) , but as lifetimes longer than 100s were capped because 
 of the technical limitations on for how long an experiment can continue , 
 this should be considered a lower limit . therefore , there is an indication 
 that the e286c mutant may have a longer lifetime for proton uptake / release . behavior was observed 
 in only 4% of the active vesicles . the relative frequency ( in percentage ) 
 of cytochrome bo3 entering the leak state 
 or stalling phase within the 
 time resolution of the experiment as a function of ph . ( a ) 
 the average frequencies that a ph is encountered during a 2.6 
 s period of the time trace ( i.e. , the time resolution ) ; ( b , c ) the 
 distribution of the ph formed just before entering the leak 
 state or stalling phase , respectively ; ( d , e ) : the relative frequency 
 of cytochrome bo3 entering the leak state 
 or stalling phase , respectively , as a function of ph , which 
 was calculated by dividing the frequencies in ( b ) and ( c ) by those 
 in ( a ) for each independent experiment ; the error bars show the standard 
 error of the mean from 4 independent experiments . the negative ph 
 and negative proton release rates ( acidification ) have been converted 
 to the absolute ( positive ) values . the ph is the intravesicular 
 ph minus the ph of the bulk solution ( constant at 7.4 ) . we next examined whether the existence of the leak 
 state may have 
 a potential regulatory role . a possible cause that triggers cytochrome bo3 into the leak state is a thermodynamically 
 limiting pmf , i.e. , high ph and/or high membrane potential . figure 5 shows that the leak 
 states may be initiated at all ph ( horizontal axis in the heat 
 map ) , and many leaking events started at low ph . however , the 
 relative frequency shown in figure 5 is the frequency observed for the total population 
 of active vesicles , which does not take into account that the majority 
 of active vesicles never reached a high ph or the fact that 
 high ph are only obtained for a relatively short time periods 
 ( figure 6a ) . figure 6d shows that by normalizing 
 the frequency of the leak state against the relative occurrence of 
 a ph , the leak states is more likely to be initiated at higher 
 ph . the result of kendall tau rank correlation test shows that 
 the relative frequency of entering a leak state from all repeated 
 experiments is indeed positively correlated to the ph ( p < 0.01 ,  = 1 ) . the same analysis shows that 
 the probability of entering a stalling phase is similarly correlated 
 to the ph ( figure 6e ) . thus , our data suggest that cytochrome bo3 might have evolved two mechanisms to control excessive 
 ph and either halts proton translocation or enters a proton 
 leak state , dissipating the formed pmf . as mentioned above , the e286c 
 mutant exhibits a longer lifetime of proton translocation . as the 
 relative frequencies of the leak and stalling states of cytochrome bo3 are dependent on the ph ( figure 6 ) , the longer lifetime 
 could thus indirectly be due to the slower proton uptake / release activity 
 of e286c cytochrome bo3 compared to wt . proteoliposomes are ideally suited for this as it 
 has long been recognized that due to small volume in proteoliposomes , 
 proton transport alone can build up a membrane potential . to probe the influence of the membrane potential , 
 the same experiment and analysis were performed on the wild - type cytochrome bo3 in the presence of a potassium ion carrier , 
 valinomycin , which will significantly reduce or eliminate the membrane 
 potential , while retaining the ph . by comparing the results 
 of cytochrome bo3 alone and cytochrome bo3 with valinomycin in figure 5 , it is clear that valinomycin lowers the 
 leaking rate ( see also figure s5 ) . on average , the presence of valinomycin reduced the leaking rate by 60% , confirming 
 that a significant membrane potential is present and thus that proton 
 transfer by cytochrome bo3 contributes 
 to the formation of both terms of the pmf in our single - enzyme experiments 
 ( ph and membrane potential ) . the relative frequency , however , 
 with which cytochrome bo3 enters the leak 
 state is not reduced by the presence or absence of valinomycin . as 
 without valinomycin , the relative frequency of entering the leak state is correlated to the ph in the presence of valinomycin ( figure 
 s6 , see si for a further discussion on 
 the effect of valinomycin on the proton transport activity of cytochrome bo3 ) . these observations suggest that entering 
 the leak state may be primarily triggered by the ph , though 
 we can not fully exclude that a larger membrane potential can also 
 trigger a leak state . in contrast , the rate of the proton back flow 
 once a leak state is obtained seems to be driven by both elements 
 of the pmf ( figure 5 ) suggesting that the leak state is due to a protein conformation 
 that allows protons to freely flow backward , in a fashion similar 
 to a proton channel . we conclude that a long - lifetime 
 leak state exists in cytochrome bo3 during 
 continuous enzyme turnover , which 
 allows the back flow of protons . the leak state and the stalling in 
 enzyme activity may act as a safety fuse that is triggered by excessive 
 ph during respiration in e. coli . previous 
 study shows that ros production in mitochondria may be much more sensitive 
 to ph than to membrane potential . although seemingly inefficient in terms of energy conservation , 
 proton leakage in small subpopulations of hcos in a living organism 
 might help to alleviate respiratory pressure and factors related to 
 ros production . the existence of the long - lifetime proton leak state 
 and the stalling phase may provide an unprecedented insight into the 
 proton pumping mechanism and its regulation in hcos . deciphering the 
 precise role of proton leakage of proton transporters as well as its 
 dependence on regulatory inputs and point mutations by assays like 
 these will pave the way for controlling transport related diseased 
 states . wild - type 
 and e286c cytochrome bo3 were purified 
 from escherichia coli c43 strains with the bo3 gene knocked out from chromosome , where proteins 
 were expressed from pet plasmids containing the corresponding gene 
 sequences . cytochrome bo3 concentration was determined by its soret 
 band at 409 nm (  = 188 mm cm ) . protein was reconstituted following a method published previously , except 20 mm mops buffer was used instead of 
 hepes . before bio - beads were added , the ph sensitive dye , 8-hydroxypyrene-1,3,6-trisulfonic 
 acid trisodium salt ( hpts ) , was added to a final concentration of 
 5 mm . the lipid mixture used for the reconstitution was e. 
 coli polar lipid extract ( avanti polar lipids , inc . , alabama , 
 u.s.a . ) mixed with the following : 1% ( w / w ) ubiquinone-10 and 0.4% 
 ( w / w ) atto-633 labeled dope ( atto - tec gmbh , germany ) . the protein - to - lipid 
 weight percentage added for the reconstitution is 0.2% . insoluble 
 materials , possibly denatured protein , were removed by low - speed centrifugation 
 at 17  000 rcf for 5 min at the end of the reconstitution . standard 
 bca and schaffner & weissmann assays were both used to quantify 
 the amount of protein that is actually reconstituted into lipid vesicles , 
 and the loss of lipid during reconstitution was also quantified by 
 the absorption of the atto633 labeled lipid . the results show that 
 the final protein to dry lipid ratio ( w / w ) is 0.073  0.004% . 
 to estimate the average number of bo3 complexes 
 in a single vesicle , the following values were used : the protein / lipid 
 weight ratio ( 0.073% ) , the size of the vesicle ( 70 nm ) , the thickness 
 of the lipid bilayer ( 5 nm ) , the average surface area of the lipid 
 headgroup ( 65  ) , the molecular weight of the protein 
 ( 144 kda ) , and the average molecular weight of the lipid ( 760 ) . under 
 the assumption that a lipid vesicle is a double - layered sphere with 
 an outer diameter of 70 nm and an inner diameter of 70  2 
  5 = 60 nm , one can calculate the number of lipid molecules 
 in a vesicle . using the protein / lipid ratio , one can further calculate , 
 on average , how many protein complexes are present in a vesicle . dynamic 
 light scattering data showed that ( proteo)liposomes in this study 
 were highly monodispersed and are about 70 nm in diameter ( figure s7 ) . the experimental 
 setup was refined from a method reported previously to enable single - molecule measurements . template - stripped 
 gold electrodes with highly smooth transparent gold layers on glass 
 substrates were functionalized with a 6-mercapto-1-hexanol ( 6-mh ) 
 self - assembled monolayer ( sam ) . they were prepared by the following 
 procedure : 30-nanometer gold layers were thermally evaporated on polished 
 silicon wafers ( idb technologies ltd . , u.k . ) with an edwards auto 
 306 evaporator ( edwards , u.k . ) standard - thickness glass coverslips were 
 then glued to the gold layer with a low - fluorescence epoxy glue ( 3012fl , 
 epoxy technology , inc . , massachusetts , u.s.a . ) . after the glue cured , 
 coverslips were carefully removed from the silicon wafers , during 
 which the gold layer was stripped off the wafers , retaining the high 
 smoothness of the silicon wafer template . the sam layer was made by 
 incubating the gold electrodes in 1 mm 6-mh solution in water overnight 
 at 20 c . the gold electrode is a part of a custom - built electrochemical 
 cell that can be used with an inverted epi - fluorescence microscope 
 ( eclipse ti  u , nikon instruments , u.k . ) . after assembling the 
 electrochemical cell , ( proteo)liposomes were sparsely absorbed to 
 the gold electrode by incubating the gold surface with a diluted suspension 
 ( 2 g / ml lipid ) for 30 min at 20 c followed by a thorough 
 washing step to remove unbound ( proteo)liposomes and nonencapsulated 
 hpts dye . we note that the experiments are conducted under ambient 
 conditions and in ensemble experiments we have previously shown that 
 this ensures that cytochrome bo3 remains 
 saturated with oxygen throughout the experiment . images and image series were recorded using a 60 
 oil immersion objective ( n.a . = 1.40 , nikon instruments , u.k . ) , and 
 an andor zyla 5.5 scmos camera system ( andor technology ltd . , u.k . ) . fluorescence filters used in this study were from chroma technology 
 corporation ( vermont , u.s.a . ) : ( 1 ) d470/20x , excitation of one of 
 the hpts channels ; ( 2 ) d410/30x , for excitation in another hpts channel ; 
 ( 3 ) hq535/48 m , emission of both hpts channels ; ( 4 ) 500dcxr , dichroic 
 mirror for both hpts channels ; ( 5 ) hq620/60x , excitation of the atto633 
 fluorescence ; ( 6 ) hq700/75 m , emission of the atto633 fluorescence ; 
 ( 7 ) q660lp , dichroic mirror for atto633 fluorescence . the magnification 
 is 60 ( objective )  1.5 ( relay lens ) = 90 times . the exposure 
 time is 1 s for each channel , and the frame rate is 2.6 s , including 0.6 s for rotating the filter cubes . a potentiostat ( chi604c , ch instruments , inc . , texas , u.s.a . ) 
 was 
 used to control the potential between the working electrode ( the gold 
 electrode with lipid vesicles ) and a ag / agcl / sat . kcl ( 199 mv vs standard 
 hydrogen electrode , she ) reference electrode ( radiometer analytical 
 sas , france ) . a platinum wire was used as the counter electrode . when 
 the potential is applied to the gold electrode , the ubiquinone pool 
 in the vesicles lipid bilayer is electrochemically reduced 
 and after diffusion through the vesicle , donates electrons to cytochrome bo3 , initiating oxygen reduction and proton uptake / release . after a given amount of time ( typically 30 or 100 s ) 
 the potentiostat cell was switched on to maintain a constant potential 
 of 0.2 v vs she for 100 s. this potential is optimal based 
 on previous results . the potentiostat 
 was then switched off again but the image recording would continue 
 for certain amount of time ( typically 100 or 150 s ) . when the electrochemical 
 potential is terminated , the proton uptake / release activity is found 
 to halt within the time resolution of this study and , thus , the enzyme 
 activity is tightly controlled by the potentiostat . there is a minimum 
 of 15 min waiting period between each recording to allow the ph 
 and membrane potential to dissipate , and the fields of view of different 
 recording sessions do not overlap . typically 5 recordings were done 
 on each gold electrode due to the limit of their available surface 
 area . each experimental system ( wild - type bo3 , e286c mutant and valinomycin ) contains data collected from 
 different batches of preparation and each batch contains recordings 
 from multiple electrodes . the numbers of independent lipid vesicles 
 analyzed in each of the three experimental system described in the 
 main text were on the scale of 19  00030  000 , 
 of which about 3000 vesicles exhibit proton uptake / release . cytochrome bo3 was reconstituted 
 using the same protocol as above and valinomycin was added to a final 
 concentration of 10 m with the lipid concentration adjusted 
 to 4.4 mg / ml . the valinomycin was incubated 
 with ( proteo)liposomes for 15 min before further dilution to 2 g / ml 
 lipid and absorption onto the gold electrode , as above . however , after 
 unbound ( proteo)liposomes and nonencapsulated hpts dye were removed 
 by washing , a new valinomycin / liposome mixture ( 10 m and 4.4 
 mg / ml , respectively ; liposomes were without hpts or cytochrome bo3 ) was added to the solution to replenish the 
 loss of valinomycin during the incubation and washing and keep the 
 ratio of lipid and valinomycin relatively constant . since the liposomes 
 added here do not contain any fluorescent dye or fluorescently labeled 
 lipid , they do not interfere with the image recording . before the start of an experiment a single 
 fluorescence image was taken to obtain the atto-633 fluorescence intensity 
 of individual ( proteo)liposomes containing 0.4% ( w / w ) atto-633 labeled 
 dope . this image was used to calculate the diameter of the ( proteo)liposomes 
 with a method similar to that previously published . dynamic light scattering ( zetasizer nano zs , malvern instruments 
 ltd . , u.k . ) data were used to establish a calibration curve between 
 atto-633 fluorescence and the diameter of the vesicles . figure s8 shows that the conversion from atto 
 fluorescence to diameter does not distort the size distribution of 
 the lipid vesicles . fluorescent image time - traces were recorded 
 alternating between the excitation filters for hpts fluorescence . image series were registered and corrected for drifting using imagej 
 stackreg plug - in . all subsequent analysis 
 was performed in matlab r2012a ( mathworks , massachusetts , u.s.a . ) 
 using user - written code . lipid vesicles were identified on each frame 
 of the image series and a two - dimensional gaussian function was used 
 to fit each vesicle hpts fluorescence image . using user - written code , 
 comprehensive and automatic quality checking was carried out for each 
 fitting on every frame to eliminate conglomerates , close - by vesicles , 
 vesicle desorption during recording and vesicles on the edge of images . the code was written assuming the following : ( 1 ) single vesicles will 
 appear as circular bright spots on the image ; ( 2 ) because the bright 
 spots are sparsely distributed on the images , the majority of them 
 are single vesicles ; ( 3 ) a cluster of vesicles will appear either 
 noncircular or much larger . an automated image analysis was performed 
 with matlab to remove noncircular spots or spots larger than expected 
 for single vesicles . as the vesicles are sparsely adsorbed , an accurate 
 value for the background intensity can be determined . vesicles are 
 then identified by the matlab code by finding pixels above certain 
 threshold intensity . then a 12-by-12 ( pixel ) local image ( 0.84 
  0.84 m ) around the bright pixel was fitted to a 2-dimension 
 gaussian function . the fitting returns 4 parameters ( baseline , amplitude , 
 x - spread and y - spread ) and the fitting error at each pixel . these 
 results are used to reject bright spots with the following characteristics : 
 ( 1 ) fitting error above 20% ( comparing to the original intensity data ) 
 at any pixel ; ( 2 ) fitting error above 10% overall ( in the 12-by-12 
 area ) ; ( 3 ) circularity below 0.8 ; ( 4 ) fwhm is below 35 nm (= 0.5 pixel ) 
 or above 175 nm (= 2.5 pixel ) ; ( 5 ) amplitude above background is below 
 1000 au ( arbitrary unit , background is about 3000 au with a standard 
 deviation about 150 au ) ; ( 6 ) baseline above 10  000 au ( this 
 is usually concurrent with a large fwhm ) ; ( 7 ) too close to the edge 
 of image ( 6 pixel ) ; ( 8) having another bright spot within 700 nm ; 
 ( 9 ) the fitted center is more than 2 pixel away from the brightest 
 pixel ( this is usually concurrent with a large fwhm ) ; ( 10 ) in addition , 
 after all images in a movie are fitted , if any of the above criteria 
 is not satisfied in more than 30% of the frames of the movie , the 
 vesicle is rejected entirely . the above criteria were chosen because 
 they result in picking circular and separated bright spots with a 
 typical size that is consistent with single vesicles . the ratio 
 of the fluorescence intensity of hpts with the two excitation 
 filters was used to calculate the ph inside the individual ( proteo)liposome 
 at the time of measurement . this calibration was done 
 by measuring single ( proteo)liposomes hpts fluorescence in 
 the same procedure as described above at different ph values . different 
 ph points were achieved by changing the bulk solution s ph 
 and adding 100 ng / ml nigericin and 100 ng / ml gramicidin to equilibrate 
 the intravesicular ph with the outside . because of the presence of 
 gramicidin and nigericin , the intravesicular ph equals the ph of the 
 bulk solution , and the bulk solution ph was measured . the fluorescence images for ph - fluorescence calibration were taken 
 using the same procedure as the movies . the fluorescence data and 
 ph were fitted to a henderson  hasselbalch equation and the 
 parameters obtained were used to convert hpts ratiometric fluorescence 
 to intravesicular ph during turnover of cytochrome bo3 . the fitting results are given in supporting information ( figure s10 and table s1 ) . the number 
 of protons taken / released was then calculated based on the diameter 
 of lipid vesicle , the ph change and the buffering effect of mops buffer , 
 hpts and the lipids . we note that the obtained time traces , such as 
 shown in figure 2 , 
 are qualitatively identical irrespective of whether ph or cumulative 
 number of transported protons is plotted ( compare figure 2 with figure s1 ) . to calculate the cumulative number of transported protons , 
 the concentrations of mops buffer and hpts encapsulated within the 
 lipid vesicles were assumed to be the same as the bulk solution . the 
 buffering capacity of lipids was determined empirically by a titration 
 beforehand while the lipid surface area of single vesicles was calculated 
 from the vesicle size . each ( proteo)liposome s 
 change in the ph and the number of taken / released protons were then 
 calculated and stored . the sections of time traces recorded 
 after the applied potential 
 was switched off were fitted with an exponential decay function . the 
 sections of the time traces within the potential window were then 
 analyzed using user - written code to detect the proton taken up / released 
 and leaking events . this automatic analysis is based on the hypothesis 
 that a correlation between the ph / number - of proton taken up / released 
 and time indicates either an active proton uptake / release or leaking 
 event . it consists of fitting the data to models of one or multiple 
 joint lines and , for each line ( segment ) , the calculation of kendall 
 tau rank correlation coefficient . in order to prevent overfitting 
 of the trace by multiple lines , the fitting would start with a single 
 linear regression ( a single segment ) . if the standard deviation of 
 the residual was less than 110% of the standard deviation of the trace , 
 the procedure was stopped and the kendall tau value determined . the 
 standard deviation of the trace is independently determined using 
 the data prior to the application of the potential . it is assumed 
 that the intravesicular ph before the applying a potential is constant 
 and that the noise level is consistent during the entire trace . if 
 the standard deviation of the residual of the segment is higher than 
 110% , the data is fitted to two joined segments ( i.e. , the start of 
 the second segment joins with the end of the first segment ) , where 
 an iterative process is used to determine , based on the best fit , 
 at which time - point the two segments are connected . if any of the 
 segments still has a residue with a standard deviation above 110% , 
 this process is continued . to further check if the use of multiple 
 segments is statistically 
 justified , adjusted r values are determined 
 for fits using one , two or more segments , and the fit with the highest 
 adjusted r value is used . adjusted r takes into account the degrees of freedom 
 in relation to the number of fitted parameters . p - values were calculated as part of the kendall test on the resolved 
 sections from the model fitting and only the sections of time traces 
 with p - values below the threshold of 0.05 ( 95% confidence 
 level ) were accepted as proton uptake / release or leaking events . proton 
 uptake / release or leaking rates are taken from the slopes of the segments . the data was analyzed with higher and lower confidence levels and 
 was found to be qualitatively identical . finally , a leak event is 
 defined as a change in the sign of a slope for two adjoining segments , 
 where it is assumed that the first segment is due to active proton 
 uptake / release .
heme - copper oxidases ( hcos ) are key 
 enzymes in prokaryotes and 
 eukaryotes for energy production during aerobic respiration . 
 they 
 catalyze the reduction of the terminal electron acceptor , oxygen , 
 and utilize the gibbs free energy to transport protons across a membrane 
 to generate a proton ( ph ) and electrochemical gradient termed 
 proton motive force ( pmf ) , which provides the driving force for the 
 adenosine triphosphate ( atp ) synthesis . 
 excessive pmf is known to 
 limit the turnover of hcos , but the molecular mechanism of this regulatory 
 feedback remains relatively unexplored . 
 here we present a single - enzyme 
 study that reveals that cytochrome bo3 from escherichia coli , an hco closely homologous 
 to complex iv in human mitochondria , can enter a rare , long - lifetime 
 leak state during which proton flow is reversed 
 . the probability of 
 entering the leak state is increased at higher ph . by rapidly 
 dissipating the pmf , 
 we propose that this leak state may enable cytochrome bo3 , and possibly other hcos , to maintain a suitable 
 ph under extreme redox conditions .