Mapping protein dynamics in catalytic intermediates of the ...several water molecules observed in...

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Mapping protein dynamics in catalytic intermediates of the redox-driven proton pump cytochrome c oxidase Laura S. Busenlehner*, Lina Salomonsson , Peter Brzezinski , and Richard N. Armstrong* ‡§ Departments of *Biochemistry and Chemistry, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146; and Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden Edited by Judith P. Klinman, University of California, Berkeley, CA, and approved August 18, 2006 (received for review February 21, 2006) Redox-driven proton pumps such as cytochrome c oxidase (CcO) are fundamental elements of the energy transduction machinery in biological systems. CcO is an integral membrane protein that acts as the terminal electron acceptor in respiratory chains of aerobic organisms, catalyzing the four-electron reduction of O 2 to H 2 O. This reduction also requires four protons taken from the cytosolic or negative side of the membrane, with an additional uptake of four protons that are pumped across the membrane. Therefore, the proton pump must embody a ‘‘gate,’’ which provides alternating access of protons to one or the other side of the membrane but never both sides simultaneously. However, the exact mechanism of proton translocation through CcO remains unknown at the molec- ular level. Understanding pump function requires knowledge of the nature and location of these structural changes that is often difficult to access with crystallography or NMR spectroscopy. In this paper, we demonstrate, with amide hydrogendeuterium ex- change MS, that transitions between catalytic intermediates in CcO are orchestrated with opening and closing of specific proton pathways, providing an alternating access for protons to the two sides of the membrane. An analysis of these results in the frame- work of the 3D structure of CcO indicate the spatial location of a gate, which controls the unidirectional proton flux through the enzyme and points to a mechanism by which CcO energetically couples electron transfer to proton translocation. conformational change mass spectrometry hydrogendeuterium exchange T wo proton-conducting pathways, composed of polar residues and bound water molecules leading from the cytosol to the catalytic site of cytochrome c oxidase (CcO) from Rhodobacter sphaeroides, have been identified through biochemical and struc- tural analysis (refs. 1–4; Fig. 1A). One pathway, the K pathway, provides 1–2 protons to reduce the catalytic site. The second pathway, the D pathway, supplies the remaining substrate pro- tons plus four protons to be pumped across the membrane. Therefore, the D pathway must contain a branching point from which protons are distributed either to the catalytic site or to an outlet for the pumped protons. This branching point is thought to be at or near E286, which resides at the end of the D pathway (1, 8). After E286, the pathway for proton transfer to the catalytic site or to the exit channel is unclear but may involve transient hydrogen-bonded water chains that translocate sub- strate protons to the catalytic site (9–12). The exit channel for pumped protons is proposed to begin in the area around the D ring propionates of hemes a and a 3 (1, 12–15). It is anticipated that control of proton distribution involves local structural changes near these propionates that are linked to conforma- tional changes involving E286 and surrounding residues (1, 12, 16, 17). The location of residues involved in accepting protons and the organization of water molecules are crucial for deter- mining the mechanism of redox-coupled proton translocation. Three-dimensional structures of various oxidases provide a framework for functional analysis but do not define how these enzymes coordinate proton distribution during turnover. In this paper, we report the detection of distinct redox-linked conforma- tional transitions in the catalytic cycle of CcO by monitoring amide hydrogendeuterium (HD) exchange kinetics along the protein backbone by MS (18). Briefly, catalytic intermediate states of CcO were pulsed with deuterium oxide (D 2 O) at pH 7.0 for varying times (15 s to 6 h), quenched with acid to stop in-exchange, and digested with either pepsin or Rhizopus newlase proteases. The resulting peptides were separated by HPLC, and deuterium incorporation was determined as a function of time by MS. The kinetics of HD exchange monitors differences in structure and dynamics among discrete intermediate states and not the actual transitions between them. The four redox states of CcO that are sufficiently stable for analysis include the oxidized state (O), the four-electron reduced state (R), the ‘‘peroxy’’ intermediate (P m ), and the ‘‘ferryl’’ inter- mediate (F) (Fig. 1B). The rates at which amide HD exchange occurs depends on many factors (19, 20). Despite this observation, the fast phase of HD exchange occurring in the first 15 s (k 4 min 1 ) generally reports on the relative solvent accessibility of amide protons, whereas the intermediate and slow phases reflect changes in protein dynamics (19, 20). Results and Discussion Proton-Uptake Pathways. The K pathway, formed, in part, by residues K362, T359, S365, and Y288 (1), provides one to two protons during the O3R transition (2, 21–23). In the O state, a peptide containing residues 354 –366 (Fig. 2), which embodies most of the K pathway (Fig. 1 A), is in a solvent-inaccessible conforma- tion as shown by the low percentage of amide hydrogens that exchange in the fast phase (18% deuterium incorporation within 15 s) (Fig. 3A). However, after full reduction (R state), the K pathway opens to solvent, exposing the amide hydrogens to base- catalyzed exchange that results in increased incorporation of deu- terium along the backbone (63% within 15 s). Although only two water molecules within the channel are observed in the structure of oxidized CcO (Fig. 1 A), it is clear that to form R, the K pathway must create a hydrogen-bonded chain for efficient proton transfer (1). These data indicate that proton uptake through the K pathway for reduction of the catalytic site involves a specific conformational transition that allows water from the bulk solution to participate in the translocation. Upon formation of the P m state and further to the F state, the peptide containing the K pathway residues has restricted access to solvent (Fig. 3A), consistent with its role exclusively in the reductive half of the cycle (O3R) (2, 22, 23). These results are also Author contributions: P.B. and R.N.A. designed research; L.S.B. and L.S. performed research; L.S. contributed new reagentsanalytic tools; L.S.B., P.B., and R.N.A. analyzed data; and L.S.B., P.B., and R.N.A. wrote the paper. The authors declare no conflict of interest. This article is a PNAS direct submission. Abbreviations: CcO, cytochrome c oxidase; HD, hydrogendeuterium; O, oxidized state; R, reduced state; Pm, peroxy intermediate; F, ferryl intermediate. § To whom correspondence should be addressed. E-mail: [email protected]. © 2006 by The National Academy of Sciences of the USA 15398 –15403 PNAS October 17, 2006 vol. 103 no. 42 www.pnas.orgcgidoi10.1073pnas.0601451103 Downloaded by guest on November 11, 2020

Transcript of Mapping protein dynamics in catalytic intermediates of the ...several water molecules observed in...

Page 1: Mapping protein dynamics in catalytic intermediates of the ...several water molecules observed in the crystal structure form a hydrogen-bonded network that connects hemes a a 3 and

Mapping protein dynamics in catalyticintermediates of the redox-drivenproton pump cytochrome c oxidaseLaura S. Busenlehner*, Lina Salomonsson†, Peter Brzezinski†, and Richard N. Armstrong*‡§

Departments of *Biochemistry and ‡Chemistry, Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146;and †Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden

Edited by Judith P. Klinman, University of California, Berkeley, CA, and approved August 18, 2006 (received for review February 21, 2006)

Redox-driven proton pumps such as cytochrome c oxidase (CcO) arefundamental elements of the energy transduction machinery inbiological systems. CcO is an integral membrane protein that actsas the terminal electron acceptor in respiratory chains of aerobicorganisms, catalyzing the four-electron reduction of O2 to H2O.This reduction also requires four protons taken from the cytosolicor negative side of the membrane, with an additional uptake offour protons that are pumped across the membrane. Therefore, theproton pump must embody a ‘‘gate,’’ which provides alternatingaccess of protons to one or the other side of the membrane butnever both sides simultaneously. However, the exact mechanism ofproton translocation through CcO remains unknown at the molec-ular level. Understanding pump function requires knowledge ofthe nature and location of these structural changes that is oftendifficult to access with crystallography or NMR spectroscopy. In thispaper, we demonstrate, with amide hydrogen�deuterium ex-change MS, that transitions between catalytic intermediates in CcOare orchestrated with opening and closing of specific protonpathways, providing an alternating access for protons to the twosides of the membrane. An analysis of these results in the frame-work of the 3D structure of CcO indicate the spatial location of agate, which controls the unidirectional proton flux through theenzyme and points to a mechanism by which CcO energeticallycouples electron transfer to proton translocation.

conformational change � mass spectrometry � hydrogen�deuterium exchange

Two proton-conducting pathways, composed of polar residuesand bound water molecules leading from the cytosol to the

catalytic site of cytochrome c oxidase (CcO) from Rhodobactersphaeroides, have been identified through biochemical and struc-tural analysis (refs. 1–4; Fig. 1A). One pathway, the K pathway,provides 1–2 protons to reduce the catalytic site. The secondpathway, the D pathway, supplies the remaining substrate pro-tons plus four protons to be pumped across the membrane.Therefore, the D pathway must contain a branching point fromwhich protons are distributed either to the catalytic site or to anoutlet for the pumped protons. This branching point is thoughtto be at or near E286, which resides at the end of the D pathway(1, 8). After E286, the pathway for proton transfer to thecatalytic site or to the exit channel is unclear but may involvetransient hydrogen-bonded water chains that translocate sub-strate protons to the catalytic site (9–12). The exit channel forpumped protons is proposed to begin in the area around the Dring propionates of hemes a and a3 (1, 12–15). It is anticipatedthat control of proton distribution involves local structuralchanges near these propionates that are linked to conforma-tional changes involving E286 and surrounding residues (1, 12,16, 17). The location of residues involved in accepting protonsand the organization of water molecules are crucial for deter-mining the mechanism of redox-coupled proton translocation.

Three-dimensional structures of various oxidases provide aframework for functional analysis but do not define how theseenzymes coordinate proton distribution during turnover. In this

paper, we report the detection of distinct redox-linked conforma-tional transitions in the catalytic cycle of CcO by monitoring amidehydrogen�deuterium (H�D) exchange kinetics along the proteinbackbone by MS (18). Briefly, catalytic intermediate states of CcOwere pulsed with deuterium oxide (D2O) at pH 7.0 for varying times(15 s to 6 h), quenched with acid to stop in-exchange, and digestedwith either pepsin or Rhizopus newlase proteases. The resultingpeptides were separated by HPLC, and deuterium incorporationwas determined as a function of time by MS. The kinetics of H�Dexchange monitors differences in structure and dynamics amongdiscrete intermediate states and not the actual transitions betweenthem. The four redox states of CcO that are sufficiently stable foranalysis include the oxidized state (O), the four-electron reducedstate (R), the ‘‘peroxy’’ intermediate (Pm), and the ‘‘ferryl’’ inter-mediate (F) (Fig. 1B). The rates at which amide H�D exchangeoccurs depends on many factors (19, 20). Despite this observation,the fast phase of H�D exchange occurring in the first 15 s (k � 4min�1) generally reports on the relative solvent accessibility ofamide protons, whereas the intermediate and slow phases reflectchanges in protein dynamics (19, 20).

Results and DiscussionProton-Uptake Pathways. The K pathway, formed, in part, byresidues K362, T359, S365, and Y288 (1), provides one to twoprotons during the O3R transition (2, 21–23). In the O state, apeptide containing residues 354–366 (Fig. 2), which embodies mostof the K pathway (Fig. 1A), is in a solvent-inaccessible conforma-tion as shown by the low percentage of amide hydrogens thatexchange in the fast phase (18% deuterium incorporation within15 s) (Fig. 3A). However, after full reduction (R state), the Kpathway opens to solvent, exposing the amide hydrogens to base-catalyzed exchange that results in increased incorporation of deu-terium along the backbone (63% within 15 s). Although only twowater molecules within the channel are observed in the structure ofoxidized CcO (Fig. 1A), it is clear that to form R, the K pathwaymust create a hydrogen-bonded chain for efficient proton transfer(1). These data indicate that proton uptake through the K pathwayfor reduction of the catalytic site involves a specific conformationaltransition that allows water from the bulk solution to participate inthe translocation. Upon formation of the Pm state and further to theF state, the peptide containing the K pathway residues has restrictedaccess to solvent (Fig. 3A), consistent with its role exclusively in thereductive half of the cycle (O3R) (2, 22, 23). These results are also

Author contributions: P.B. and R.N.A. designed research; L.S.B. and L.S. performed research;L.S. contributed new reagents�analytic tools; L.S.B., P.B., and R.N.A. analyzed data; andL.S.B., P.B., and R.N.A. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS direct submission.

Abbreviations: CcO, cytochrome c oxidase; H�D, hydrogen�deuterium; O, oxidized state; R,reduced state; Pm, peroxy intermediate; F, ferryl intermediate.

§To whom correspondence should be addressed. E-mail: [email protected].

© 2006 by The National Academy of Sciences of the USA

15398–15403 � PNAS � October 17, 2006 � vol. 103 � no. 42 www.pnas.org�cgi�doi�10.1073�pnas.0601451103

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in agreement with data from small molecule inhibitors of protonuptake that indicate differences in water access with changes in theredox state of CcO (24).

Unlike the K pathway, the D pathway contains a number ofcrystallographically resolved water molecules that connect thecytoplasm of subunit I to E286 (1), suggesting the existence ofa preorganized proton conducting ‘‘wire’’ (Fig. 1 A). Despite thepresence of these bound waters, amide H�D exchange kineticsof several peptides spanning the D pathway reveal that watermolecules in the channel either do not exchange rapidly withbulk solvent or that base-catalyzed H�D exchange is unfavorablein all intermediate states (Fig. 3 B–E). In the O state, one peptidethat displays moderate deuterium incorporation at longer incu-bation times is 282–292 (Fig. 2), which contains the gatingresidue E286 and the H284-Y288 covalent cross-link.¶ In the Rstate, this peptide incorporates less deuterium over 6 h com-pared with O (Fig. 3B). This result indicates that when the Kpathway is open to reduce the catalytic site, the terminal regionof the D pathway near the catalytic site becomes less dynamicand more restrictive to water access. This change may reflectformation of a barrier limiting protonic contact between the twopathways. Further, upon formation of the Pm and F intermedi-ates, deuterium exchange into peptide 282–292 is reversed anddisplays kinetic profiles similar to the O state. Although nospecific data on the proposed E286 proton gate can be obtained,it is clear that the backbone between residues 282–292 is able toundergo redox-dependent conformational changes.

Interestingly, a cytosolic loop (peptide 540–551) at the en-

¶The H284-Y288 cross-linked peptide (282–292) was observed in all intermediate states;however, a small population of a noncross-linked peptide corresponding to residues283–304 was observed consistently in the O, R, and Pm states, but not the F state. The H�Dexchange kinetic profiles for the O and Pm states of peptide 283–293 without the cross-link(generated by subtraction of 293–304) are similar to those of 282–292 with the cross-link,as shown in Fig. 6, which is published as supporting information on the PNAS web site.

Fig. 1. Proton transfer pathways and the catalytic reaction cycle for O2

reduction. (A) The D and K pathways are indicated with black and blue solidlines, respectively, where CuA (green), heme a (yellow), CuB (light green),heme a3 (cyan), Mg2� (magenta), and H2O (red spheres) are shown. D pathwayprotons are directed to the catalytic site for O2 reduction (dashed black line)or to be pumped (dashed red line). (B) The oxidized catalytic site (O) is reducedwith two electrons and two protons from the K pathway to form (R). Dioxygenbinds to heme a3 (A), then the O-O bond is broken forming the peroxyintermediate (Pm). Transfer of an electron and a D pathway proton results information of the ferryl intermediate (F). An additional electron is transferred,along with net proton uptake through the D pathway, which re-forms O.Proton pumping (HP

�) via the D pathway occurs from Pm3F, F3O (5), andpossibly from O3R (6, 7). Tyr-288 is represented by YOH.

Fig. 2. Peptides that undergo redox-dependent conformational changes. Pep-tides that display alterations in H�D exchange kinetics of backbone amides areindicated on the structure of oxidized CcO subunits I (gray) and II (light pink). Thepeptides are colored as follows: 354–366, green; 282–292, blue; 123–135, yellow-green; 136–145, reddish-orange; 193–203, hot pink; 540–551, purple; 169–175,red; 320–340, orange; and II225–229, gold. The general location of the D and Kproton uptake channels are indicated by the black and blue arrows, respectively.E286 (blue) is shown in stick format. The kinetic profiles of deuterium incorpo-ration for each peptide are shown in Figs. 3 and 4.

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trance to the D pathway (Fig. 2) shows decreased deuteriumincorporation from O3R in the fast phase and subsequentintermediate phase (Fig. 3F). An additional overlapping peptide,540–550, also displays the same kinetic behavior and indicatesreproducibility of this change in deuterium incorporation (datanot shown). This region is proposed to act as a proton ‘‘antenna’’involving residues E548 and H549 (25–27). These results suggestthat structural organization of the loop, located outside themembrane at a distance of �40 Å from the catalytic site (1), maycreate a barrier to proton uptake through the preformed Dpathway wire when the K pathway is open. For all otherintermediate states, the kinetic profiles for 540–551 (Fig. 3F)resemble that of the O intermediate, an observation that indi-cates the conformation is specific for the R state. Although thestructure of bovine CcO has been solved in both the O and Rstates, no significant changes in conformation were observed inthe regions discussed here (28). Crystallography typically pro-vides static views of structures that do not accurately reflectchanges in protein dynamics. Clearly, more structural and bio-chemical analysis is required to understand the specific role ofthe cytosolic loop in reduction of CcO.

Proton Exit Path and the Subunit I-II Interface. Indirect evidence hasimplicated the interface between subunits I and II in the protonexit route (1, 12–15). This region is highly solvated, whereseveral water molecules observed in the crystal structure form ahydrogen-bonded network that connects hemes a�a3 and Mg2�

to the outside of the protein (1). Based on H�D exchange, oneof the most solvent-accessible peptides in this region (160–175)resides in a loop directly above E286 (Fig. 2) and harbors W172,a residue hydrogen-bonded to a heme a3 propionate thought tobe a proton acceptor in the exit route (Fig. 1 A; refs. 1, 13, 15,and 17). Peptide 160–175 is extensively exchanged with deute-rium within 2 min in the O, R, and Pm intermediates (Fig. 4A).However, the most dramatic alteration in H�D exchange occursin the F intermediate, which displays a significant decrease indeuterium content over 30 min, indicating a kinetic restriction tosolvent. Overlapping peptides indicate that the changes in H�Dexchange are located primarily between residues 169–175 (datanot shown). These results are in agreement with rapid-quenchexperiments that reported fast H2O�D2O exchange (kex � 3,000s�1) at the Mn2� site in oxidized CcO (29). The Mn2� (or Mg2�)is part of a hydrogen-bonded network; therefore, these resultssuggest that a kinetically relevant channel in this region allowswater or protons to exchange with bulk water molecules in thoseintermediate states not involved in proton pumping.

In contrast to 160–175, exchange into peptide 320–340 at thesubunit I-II interface (Fig. 2) is specifically enhanced in the Fstate and is 90% deuterated within 4 min (Fig. 4B). This peptidecontains two CuB ligands, H333 and H334. Several lines ofevidence suggest that one or more of these His residues may beinvolved in the proton exit pathway through redox-coupledchanges in CuB coordination (10, 30). The alteration in H�Dexchange into 320–340 indicates that structural changes involv-

Fig. 3. Amide H�D exchange kinetic profiles of selected K and D pathway peptides for the four intermediate states of CcO. Deuterium incorporation intoselected peptides for each intermediate state (O; red), (R; black), (Pm; blue), and (F; green) are shown. The total number (left axis) or percentage (right axis) ofdeuterium incorporated into the peptide backbone as a function of time are fit to exponential equations (Table 1, which is published as supporting informationon the PNAS web site). (A) Peptide 354–366 of subunit I contains K pathway residues. (B) Peptide 282–292 contains the D pathway residue E286 and the Y288-H284covalent cross-link. Y288 is in the K pathway and H284 is a CuB ligand. (C) Peptide 123–135 contains D132 at the entrance of the D pathway. (D) Peptide136–145contains the D pathway residue N139. (E) Peptide 193–203 contains the D pathway residue S201. (F) Peptide 540–551 of subunit I resides in a loop at the cytosolicentrance to the D pathway.

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ing these residues may control solvent access to CuB at specifictimes during catalysis. The crystal structure of CcO reveals thatresidues 320–340 also line a channel from the catalytic sitetoward bulk solvent outside of the membrane (1).

Subunit II residues II225–229 form the top of the waterchannel (Fig. 2) and are more accessible to deuterium in both thePm and F intermediates (100% incorporation by 30 min) com-pared with the O and R states (Fig. 4C). Peptide 320–340 alsoexhibits increased deuterium incorporation from R3Pm (Fig.4B). Given the proximity of II225–229 to residues 320–340 andthe observed changes in H�D exchange, it is likely that theseresidues participate in the proton exit channel. Computationalanalysis suggests that residues IIK227 and IID229 may be involvedin the proton exit pathway (31). The Pm state may play a role inorganizing the proton exit channel before pumping. The signalfor this event still is unclear, but may be related to tyrosyl radicalformation (Y288O�) at the active site.

Amide H�D exchange kinetics have led to the identification of aspecific conformational transition related to the F intermediateinvolving two regions of the protein that are connected to the samewater channel. In one region, peptide 160–175, deuterium access isrestricted, whereas H�D exchange in the second region, peptide320–340, is enhanced (Fig. 5). The kinetic profiles are reversed in

the other states. One explanation of these data is that a gate liesbetween the two regions. The F specific conformation of CcO,which leads to decreased deuterium incorporation localized toresidues 169–175, may be functionally important for proton distri-bution. Molecular dynamics simulations support a role of W172 inorganizing water molecules in the hydrophobic cavity between E286and heme a3 (12, 17). Moreover, results from studies with theW164F mutant in Paracoccus denitrificans (corresponding to W172in R. sphaeroides) implicate involvement of the equivalent residuein proton pumping (17). Taken together with the results from theH�D exchange studies, it appears that the loop surrounding W172changes its conformation during turnover to act possibly as a gatecontrolling the proton�water access through CcO. Although a smallpopulation of Pm is present in the F preparation, it is clear that theH�D exchange profiles for 160–175 and 320–340 are distinct fromPm, indicating a different conformation exists in the F state aroundthe proposed gate and proton exit pathway.

Mechanistic Implications for CcO Proton Pumps. Structural changesare expected for a proton pump in which the accessibility for protontransfer to the two sides of the membrane must change duringcatalysis. One example where such changes have been observed forcatalytic intermediate states is bacteriorhodopsin (32). However,crystal structures of oxidized and fully reduced bovine CcO do notreveal significant conformational differences between the two

Fig. 4. Amide H�D exchange kinetic profiles of selected peptides for inter-mediate states of CcO. The total number (left axis) or percentage (right axis)of deuterium incorporated into the peptide backbone as a function of timeare fit to single- or double-exponential equations as necessary (Table 2, whichis published as supporting information on the PNAS web site). (A) Peptide160–175 of subunit I is located in a loop above E286. (B) Peptide 320–340 islocated at the subunit I-II interface and contains two ligands to CuB, H333, andH334. (C) Peptide II225–229 of subunit II is located in a �-strand at the subunitI-II interface. This peptide is a subtraction of II229–235 from II225–235.

Fig. 5. Summary of redox-dependent changes in deuterium incorporation.Hemes a and a3 are represented with boxes, and the D and K pathways arerepresented with large arrows. The gate region corresponds to 169–175, wherethe solid circle indicates the gate. The proposed exit channel refers to 320–340(bottom arrow) and II225–229 (top arrow). The loop at the D pathway entrancecorresponds to 540–551. Increases and decreases in deuterium incorporationwithrespecttothepreviousstateare indicatedinredandblue, respectively. IntheR state (O3R), the K pathway experiences increased solvent access, whereas theD pathway loop and the gate region exhibit small decreases in dynamics, leadingto less deuterium incorporation. In addition, part of the exit channel shows adecrease in solvent accessibility. In the Pm intermediate (R3Pm), the K pathwaycloses to solvent, whereas the D pathway entrance loop exhibits an increase indynamics. The exit channel peptides show increases in solvent accessibility, butthe pathway is considered ‘‘closed’’ for proton pumping. Formation of the F state(Pm3F) further reduces exchange in the K pathway, presumably to allow for H�

translocation through the D pathway via a preformed proton wire. The exitchannel (320–340) opens to solvent, whereas the gate region shows a consider-abledecrease indeuteriumincorporationanddynamicsover30minofexchange.FromF3O,theexitchannelbecomesmorerestrictivetosolventandlessdynamic,resulting in decreased exchange, whereas the gate relaxes to its starting, solvent-accessible conformation.

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states (28). Here, we show that there are redox-dependent struc-tural and dynamic perturbations in Rhodobacter CcO. Amide H�Dexchange kinetics reveal conformational transitions from O3Rthat control proton uptake via the K pathway are connected tostructural changes that may block proton access to the D pathway(Fig. 5). Upon oxygen binding (R3Pm) the K pathway closes, anevent that may be linked to removal of the proposed barrier at theD pathway entrance. In addition, the exit pathway is prepared forproton pumping. Both subsequent transitions, Pm3F and F3O,are associated with structural changes that regulate proton uptakespecifically from one side of the membrane and proton release tothe other side (5, 33).

In the present study, we have been able to illustrate that thestructure of the F intermediate in the CcO reaction cycle isdifferent from that of the Pm and O states, specifically betweenthe proposed gate (residues 169–175) and proton exit channel(residues 320–340). The differences between the Pm, F, and Ostates expose conformational changes in these pathways thatare involved in CcO turnover. The Pm state prepares CcO forpumping by organizing the proton exit route. Inasmuch asproton uptake and pumping occurs before formation of F(Pm3F) and also occurs in the following transition (F3O),the ‘‘trapped’’ F intermediate represents a specific CcO con-formation that is poised for subsequent proton uptake�pumping. Our observations also indicate that the gate con-trolling this proton access may be formed, in part, by loopresidues within 169–175. This gate appears to switch accessupon formation and decay of the F state (Fig. 5), an event thatmay be controlled by the orientation and hydrogen bonding ofW172 or other loop residues. Such a gate is a central part ofa proton pump, and the results of this study provide apreviously undescribed experimental indication of its locationwithin CcO.

Concluding Remarks. Amide H�D exchange monitored by MS hasproven to be a valuable technique in probing the structural anddynamic features of proteins, now including that of the complexmembrane protein CcO. A complete understanding of enzymecatalysis requires that the conformational changes that occurduring catalysis be mapped to the structure. In this report, wedemonstrate that backbone amide H�D exchange kinetics canyield molecular insight into catalysis by exposing structuraldifferences between specific catalytic intermediates that occurduring enzymatic turnover. This application of H�D exchangeMS should open new avenues for investigation into the complexenzymatic functions of many proteins, including large, integralmembrane proteins.

Materials and MethodsGrowth of Bacteria and CcO Purification. His-tagged CcO (subunitsI–III only) was prepared from R. sphaeroides grown aerobicallywith shaking and was purified as described in ref. 34. CcO wasdemonstrated to be fully active, with turnover numbers consis-tent with those reported in ref. 34. The enzyme final concen-tration used for subsequent H�D exchange studies was 110 �Min 0.1 M Hepes, pH 7.4 supplemented with 0.1% n-dodecyl-�-D-maltoside.

Identification of Proteolytic Fragments. Pepsin or newlase proteasedigests of CcO (5:1 pepsin�CcO or 10:1 newlase�CcO wt�wt)were performed under optimized conditions (0.1 M potassiumphosphate�0.02% n-dodecyl-�-D-maltoside, pH 2.4 in H2O;8-min digestion). Peptides were separated by reversed-phaseHPLC on a microbore 1 � 50 mm C18 column with a 2–80%acetonitrile�H2O gradient (0.1 ml�min). Both mobile phasescontained 0.4% formic acid. Peptides were sequenced by usinga ThermoFinnigan (San Jose, CA) TSQ Quantum triple-quadrupole mass spectrometer in positive-ion mode by data-

dependent MS�MS collision-induced dissociation. Possible iden-tities of the peptides were determined by using massXpert (35)and were confirmed by comparison of the MS�MS spectra tofragmentation patterns generated by MS-Product (36). Proteasedigests were found to be highly reproducible under the optimizedconditions. The peptide maps of subunits I, II, and III are shownin Fig. 7, which is published as supporting information on thePNAS web site.

Amide H�D Exchange Mass Spectrometry. H�D exchange was per-formed on the O, R, Pm, and F states of CcO. The O state refersto the oxidized enzyme as purified. The R state is the four-electron reduced state obtained by incubating oxidized CcO (110�M) with excess sodium dithionite (0.2 M) under nitrogenbefore the addition of D2O to initiate exchange. The Pm state wasobtained by purging oxidized CcO (110 �M) under nitrogenbefore purging with CO gas (�99.5%) for 2 min. Each samplefor H�D exchange was allowed to incubate for 2 h, then wasexposed to air before D2O addition. The F state was prepared byadding 10 mM hydrogen peroxide to CcO (110 �M) for 2 minbefore the addition of D2O. This preparation results in 75% Fintermediate and 25% Pm intermediate. The UV-visible spectrafor each intermediate state as a function of time are shown in Fig.8, which is published as supporting information on the PNASweb site. The R, Pm, and F states are stable (�20% loss) for 6 h,2 h, and 30 min, respectively. The H�D exchange kinetics weremeasured within these limits.

Deuterium exchange was initiated by the addition of 5 �l ofCcO (67.5 �g) to 45 �l of D2O. The sample was incubated at 23°Cfor 15 s to 6 h, after which the exchange reaction was quenchedwith 50 �l of quench buffer (0.1 M potassium phosphate�0.02%n-dodecyl-�-D-maltoside, pH 2.4 in H2O; 0°C) and a subsequenttransfer to ice. After 25 s, pepsin (337 �g) or newlase (675 �g)was added, and the digestion proceeded for 8 min on ice. Allprotein samples for the H�D exchange were prepared individ-ually and were run on the same day.

The H�D exchange procedure has been described in detail inref. 37 with the differences noted below. The digested peptideswere separated over 15 min (100 �l�min) by a 2–50% acetonitrilegradient. An additional wash step with a 2-propanol-containingsolvent (50% acetonitrile�45% 2-propanol�5% H2O�0.4% for-mic acid) was included after each injection to remove detergentand undigested protein from the column. Mass spectra wererecorded on a ThermoFinnigan TSQ Quantum triple-quadrupole mass spectrometer by using positive ion electrosprayionization, essentially as described in ref. 37. Data processing wasperformed by using Finnigan Xcalibur software and MagTran(38), as outlined in ref. 37.

H�D Exchange Controls. The extent of artifactual in-exchange ofdeuterium during the quench and digestion were determined viaa zero-time control (m0%) (18). Basically, 5 �l of CcO (67.5 �g)was added to 50 �l of quench buffer at 0°C followed by additionof 45 �l of D2O. Protease was subsequently added, as described.The amount of deuterium back-exchanged for protium duringchromatography was determined by using a fully deuteratedprotein control (m100%) (18). Fully deuterated CcO was obtainedby incubating 5 �l of CcO (67.5 �g), 12.5 �l of 8 M d4-urea (2M final concentration in D2O) and 32.5 �l of D2O at 40°C for8 h. Then, 50 �l of deuterated quench buffer (0.1 M potassiumphosphate�0.02% n-dodecyl-�-D-maltoside, pD 2.4 in D2O) wasadded and incubated for an additional 2 h at 40°C. Afterincubation, the sample was digested with protease and analyzedas described. The amount of deuterium lost during HPLCfractionation was between 20–40% after normalizing to 100%deuterium incorporation, consistent with reported values (39,40). Back-exchange was calculated for each peptide in eachintermediate state and did not vary significantly with treatment.

15402 � www.pnas.org�cgi�doi�10.1073�pnas.0601451103 Busenlehner et al.

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Page 6: Mapping protein dynamics in catalytic intermediates of the ...several water molecules observed in the crystal structure form a hydrogen-bonded network that connects hemes a a 3 and

Kinetic Analysis. All kinetic traces are an average of three inde-pendent determinations for each intermediate state with eachprotease. The procedure for calculating the corrected number ofdeuterium atoms incorporated has been described in refs. 18 and37. The corrected amount of deuterium (D) incorporated in eachpeptide was plotted as a function of time and the resultingprogress curve for each peptide fit by using KaleidaGraph(Synergy Software, Reading, PA) to the sum of first-order rateterms according to Eq. 1:

D � N � �i�1

N

exp��kit� , [1]

where N is the number of amide protons that exchange at a givenrate constant, ki, during the time allowed for exchange, t (18). Thekinetic profiles were fit to single-, double-, or triple-exponentialequations as appropriate. Those amides that exchange before thefirst 15-s time point (fast phase) cannot be fit and are reported asamplitudes only with an estimated ki � 4 min�1.

Supporting Information. Additional data can be found in Tables3–5, which are published as supporting information on the PNASweb site.

This work was supported by National Institutes of Health Grants R01GM30910, F32 ES013105, T32 ES07028, and P30 ES00267 and SwedishResearch Council grants.

1. Svensson-Ek M, Abramson J, Larsson G, Tornroth S, Brzezinski P, Iwata S(2002) J Mol Biol 321:329–339.

2. Konstantinov AA, Siletsky S, Mitchell D, Kaulen A, Gennis RB (1997) ProcNatl Acad Sci USA 94:9085–9090.

3. Wikstrom M, Jasaitis A, Backgren C, Puustinen A, Verkhovsky MI (2000)Biochim Biophys Acta 1459:514–520.

4. Brzezinski P, Ädelroth P (1998) J Bioenerg Biomembr 30:99–107.5. Wikstrom M (2004) Biochim Biophys Acta 1655:241–247.6. Ruitenberg M, Kannt A, Bamberg E, Fendler K, Michel H (2002) Nature

417:99–102.7. Bloch D, Belevich I, Jasaitis A, Ribacka C, Puustinen A, Verkhovsky MI,

Wikstrom M (2004) Proc Natl Acad Sci USA 101:529–533.8. Junemann S, Meunier B, Fisher N, Rich PR (1999) Biochemistry 38:5248–

5255.9. Wikstrom M, Verkhovsky MI, Hummer G (2003) Biochim Biophys Acta

1604:61–65.10. Zheng X, Medvedev DM, Swanson J, Stuchebrukhov AA (2003) Biochim

Biophys Acta 1557:99–107.11. Cukier RI (2004) Biochim Biophys Acta 1656:189–202.12. Seibold SA, Mills DA, Ferguson-Miller S, Cukier RI (2005) Biochemistry

44:10475–10485.13. Puustinen A, Wikstrom M (1999) Proc Natl Acad Sci USA 96:35–37.14. Qian J, Mills DA, Geren L, Wang K, Hoganson CW, Schmidt B,

Hiser C, Babcock GT, Durham B, Millett F, et al. (2004) Biochemistry43:5748–5756.

15. Branden G, Branden M, Schmidt B, Mills DA, Ferguson-Miller S, BrzezinskiP (2005) Biochemistry 44:10466–10474.

16. Lubben M, Prutsch A, Mamat B, Gerwert K (1999) Biochemistry 38:2048–2056.17. Wikstrom M, Ribacka C, Molin M, Laakkonen L, Verkhovsky M, Puustinen

A (2005) Proc Natl Acad Sci USA 102:10478–10481.

18. Busenlehner LS, Armstrong RN (2005) Arch Biochem Biophys 433:34–46.19. Dharmasiri K, Smith DL (1996) Anal Chem 68:2340–2344.20. Zhang Z, Smith DL (1993) Protein Sci 2:522–531.21. Hosler JP, Shapleigh JP, Mitchell DM, Kim Y, Pressler MA, Georgiou C,

Babcock GT, Alben JO, Ferguson-Miller S, Gennis RB (1996) Biochemistry35:10776–10783.

22. Ädelroth P, Ek M, Brzezinski P (1998) Biochim Biophys Acta 1367:107–117.23. Branden M, Sigurdson H, Namslauer A, Gennis RB, Ädelroth P, Brzezinski P

(2001) Proc Natl Acad Sci USA 98:5013–5018.24. Kornblatt JA (1998) Biophys J 75:3127–3134.25. Marantz Y, Nachliel E, Aagaard A, Brzezinski P, Gutman M (1998) Proc Natl

Acad Sci USA 95:8590–8595.26. Ädelroth P, Brzezinski P (2004) Biochim Biophys Acta 1655:102–115.27. Hosler JP (2004) Biochim Biophys Acta 1655:332–339.28. Yoshikawa S, Shinzawa-Itoh K, Nakashima R, Yaono R, Yamashita E, Inoue

N, Yao M, Fei MJ, Libeu CP, Mizushima T, et al. (1998) Science 280:1723–1729.29. Florens L, Schmidt B, McCracken J, Ferguson-Miller S (2001) Biochemistry

40:7491–7497.30. Wikstrom M (2000) Biochim Biophys Acta 1458:188–198.31. Popovic DM, Stuchebrukhov AA (2005) J Phys Chem B 109:1999–2006.32. Lanyi JK (2004) Annu Rev Physiol 66:665–688.33. Faxen K, Gilderson G, Ädelroth P, Brzezinski P (2005) Nature 437:286–289.34. Mitchell DM, Gennis RB (1995) FEBS Lett 368:148–150.35. Rusconi F, Belghazi M (2002) Bioinformatics 18:644–645.36. Clauser KR, Baker P, Burlingame AL (1999) Anal Chem 71:2871–2882.37. Busenlehner LS, Codreanu SG, Holm PJ, Bhakat P, Hebert H, Morgenstern

R, Armstrong RN (2004) Biochemistry 43:11145–11152.38. Zhang Z, Marshall AG (1998) J Am Soc Mass Spectrom 9:225–233.39. Smith DL, Deng Y, Zhang Z (1997) J Mass Spectrom 32:135–146.40. Wang L, Pan H, Smith DL (2002) Mol Cell Proteomics 1:132–138.

Busenlehner et al. PNAS � October 17, 2006 � vol. 103 � no. 42 � 15403

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