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Faraday DiscussionsCite this: Faraday Discuss., 2015, 177, 547

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Spatio-temporal resolution of primaryprocesses of photosynthesis

Wolfgang Junge*

Received 24th March 2015, Accepted 25th March 2015

DOI: 10.1039/c5fd90015h

Technical progress in laser-sources and detectors has allowed the temporal and spatial

resolution of chemical reactions down to femtoseconds and A-units. In photon-

excitable systems the key to chemical kinetics, trajectories across the vibrational saddle

landscape, are experimentally accessible. Simple and thus well-defined chemical

compounds are preferred objects for calibrating new methodologies and carving out

paradigms of chemical dynamics, as shown in several contributions to this Faraday

Discussion. Aerobic life on earth is powered by solar energy, which is captured by

microorganisms and plants. Oxygenic photosynthesis relies on a three billion year old

molecular machinery which is as well defined as simpler chemical constructs. It has

been analysed to a very high precision. The transfer of excitation between pigments in

antennae proteins, of electrons between redox-cofactors in reaction centres, and the

oxidation of water by a Mn4Ca-cluster are solid state reactions. ATP, the general energy

currency of the cell, is synthesized by a most agile, rotary molecular machine. While the

efficiency of photosynthesis competes well with photovoltaics at the time scale of

nanoseconds, it is lower by an order of magnitude for crops and again lower for bio-

fuels. The enormous energy demand of mankind calls for engineered (bio-mimetic or

bio-inspired) solar-electric and solar-fuel devices.

1 Introduction

When the founders of atomic and molecular physics convened in 1927 at the 5th

Solvay Conference on “Electrons and Photons” in Brussels (see photograph in ref.1) they had established quantum mechanics, relativity and the interplay ofstochastic and conservative forces in the nano-world. Despite their immenseinsight it was probably all but evident to them howmolecular events could ever beresolved down to molecular scales of time (femto-second) and space (1 A-unit ¼0.1 nanometer). Neither was it evident that such a high resolution might beapplicable not only to small molecules but also to complex systems such asoligomers, interfaces, proteins, membranes, cells and organisms. High resolutionbecame feasible by dramatic innovations in physical instrumentation. In the

Dept. Biology & Chemistry, University of Osnabruck, R. 35/E42 Barbarastrasse 11, 49076 Osnabruck, Germany.

E-mail: [email protected]; Tel: +49-15112180743

This journal is © The Royal Society of Chemistry 2015 Faraday Discuss., 2015, 177, 547–562 | 547

http://dx.doi.org/10.1039/c5fd90015h

http://pubs.rsc.org/en/journals/journal/FD

http://pubs.rsc.org/en/journals/journal/FD?issueid=FD015177

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. View Article Online

1960s pulse lasers opened up the time domain of nanoseconds, soon followed bypico- to femtoseconds (ps–fs) in the seventies. The whole spectral range from theinfrared (IR) over the visible to the ultraviolet (UV) has been covered by pulsedphoton sources. It culminated in the free electron “laser” (rather a coherentsuper-radiation source) in the seventies, which provides femtosecond X-ray orelectron pulses. Pulse sources together with greatly improved detectors and datahandling capacities have allowed the high resolution of structure and kinetics,particularly in those physical, chemical and biological systems that can bestimulated by light.

Recent technical progress was covered in the Faraday Discussion on “EmergingPhoton Technologies for Chemical Dynamics” (FD 171). The present FaradayDiscussion (FD 177) expands this theme into “Temporally and Spatially ResolvedMolecular Science”. It aims at a rigorous exploration of the energy landscape andthe path of extremely rapid photophysical and photochemical reactions involvinga manifold of vibronic and torsional states. Two- and more-dimensional photon-echo spectroscopies have been essential to overcome the lack of spectral resolu-tion when using photon-pulses of fs-duration. Prominent topics of FD 177 havebeen quantum coherence, failure of the micro-canonical approximation, solventeffects on vibrational spectra and relaxations, and time-resolved structure anal-ysis by fs-pulses of X-rays or electrons. A common quest in most contributions toFD 177 has been the perfection of data and the outmost rigor in their theoreticalinterpretation. It has called for simple and well dened systems, mostly smallinorganic or organic molecules. The paradigms derived from the behaviour ofsimple systems – physical chemistry at its best – have provided rm grounds forbio-physical-chemists and biophysicists to address their more complex researchobjects. Although being complex in design some of nature's essential constructs,like the proteins that supply cells with energy, originated about three billionsyears ago and have been largely conserved during evolution. They are thus stan-dardized and oen as well dened as simple chemical compounds.

When dealing with these fundamental biological systems the canon of ques-tions to be asked is expanded. The key questions of physical chemistry – what,where and how fast – are to be supplemented by what for and why. The formerthree address the object, its molecular structure and evolution in time. Thequestion what for addresses the physiological role of a given construct and thequestion why whether a particular construct is exacted by laws of physics ormerely reveals nature's fancy as a legacy of evolution. In the following the relationbetween pure and biologically oriented physical chemistry is illustrated by takingoxygenic photosynthesis as an example.

2 Oxygenic photosynthesis

Oxygenic photosynthesis by cyanobacteria and plants uses sunlight and producesoxygen and biomass. Biomass serves man as food, fuel, bre and platformchemical. Early attempts to understand this process go as far back as the 18th

century. Jan Ingen-Housz in his study on vegetables noticed their “Great Power ofPurifying the Common Air in the Sunshine and of Injuring it in the Shade and atNight”.2 It was a rst appreciation of the production and re-consumption – in thereaction cycle between photosynthesis and cell respiration – of the gases that werelater coined oxygen and carbon dioxide. A rigorous spectroscopic analysis of

548 | Faraday Discuss., 2015, 177, 547–562 This journal is © The Royal Society of Chemistry 2015


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photosynthesis started half a century ago using microsecond ashes of light forexcitation. In 1961 Duysens,3 Kok,4 and Witt5 independently arrived at theconclusion that green plant photosynthesis is powered by two photosystems inseries. They drive electrons from water to NADP+. Water is oxidized by photo-system II (PSII) to yield oxygen and protons. PSII, in turn, reduces photosystem I(PSI) which reduces NADP+ to NADPH, as illustrated in Fig. 1.6 In that same yearMitchell (Nobel Prize 1978) postulated that ATP is synthesized from ADP and Pi atthe expense of a proton-motive force across the respective coupling membrane.7

The energy conserved in the chemical difference of the products NADPH/O2 (byPSII and PSI) and ATP/ADP$Pi (by ATP synthase (FOF1)) drives the reduction of CO2

to biomass.A decade later the time resolution of the primary electron transfer was

advanced into the picosecond range.8 But it took more than another decadeuntil Deisenhofer, Huber and Michel (Nobel Prize 1988) published the rststructural model of a photosynthetic reaction centre (resolution 3 A).9 It was therst ever structural model of any membrane protein. Today, structural modelsare available for all the proteins central to photosynthesis. PSI of a cyanobac-terium is resolved at a 2.5 A resolution,10 and even the larger PSI of green plantswith a molecular mass of 660 000 a.u. is resolved at 3.4 A.11 The latter hostsalmost 200 chlorophyll molecules, mostly with antennae function, plus theredox-active core with four chlorophyll- and two pheophytin molecules.12

Cyanobacterial PSII, the water–quinone oxidoreductase, has been resolvedstarting at 3.8 A13 and now at a 1.9 A resolution.14 The structure of ATP synthase(FOF1) has only partially been determined at the atomic resolution.15,16

However, its full structure is evident from low resolution data of the holo-enzyme (see ref. 6 and 17 and references therein). PSII and PSI are solid-statedevices. FOF1 is a most agile rotary machine composed of two rotary motorgenerators. FO, the ion-driven motor18,19 is mechanically coupled20 to F1, thechemical generator.15,21

The key machinery of bioenergetics (photosynthesis and respiration) datesabout three billion years ago. Its design has not changed much since then. Itjusties casting into one coherent scheme the kinetic and structural insightswhich have been elaborated on enzymes from different organisms (microorgan-isms, plants and animals).

Fig. 1 Electron and proton transfer in the coupling membrane of oxygenic photosyn-thesis. (a) Electron transfer (red arrows) and proton transfer (purple arrows) involvingphotosystems I and II (PSI and PSII), cytochrome b6f (cyt b6f), and FOF1. It produces O2,NADPH and ATP. Modified with permission from the Annual Review of Biochemistry,Volume 84. © 2015, Annual Reviews, http://www.annualreviews.org.6




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3 Excitation energy transfer

Photosynthetic reaction centres are surrounded by pigments with antennafunction, in PSII between 200 and 300 chlorophyll-molecules. Being embedded inmembrane proteins they enhance the absorption cross-section of the photo-reactive core. In chlorophyll-based reaction centres from all organisms the corehosts four plus two tetra-pyrroles, the former four being bacterio-chlorophyll andthe latter two either bacterio-pheophytin or bacterio-chlorophyll (see Fig. 2).These pigments are very closely spaced and thus strongly coupled. The core wasrst isolated in the pure form from bacteria. Starting in the eighties femtosecondspectroscopy revealed that the rst electron transfer step from bacterio-chloro-phyll to bacterio-pheophytin relaxes in picoseconds.22,23 Soon thereaer vibra-tional coherence between these pigments became evident.24 As in severalcontributions to this Faraday Discussion femtosecond excitation and multidi-mensional photon-echo spectroscopy became the clue to investigate processeswhere the micro-canonical approximation fails. An impressive series of studies onphotosynthesis has revealed quantum beats in antennae proteins (for reviews andreferences see ref. 25–27, for a recent survey of quantum biology see ref. 28).

Taking up the above biophysical question of physical necessity versus legacy ofevolution one wonders whether coherent excitation transfer is a necessaryprerequisite for the high quantum yield of photosynthesis. In a theoretical studyon a structurally well characterized bacterial antenna complex it is argued that itis rather unecessary.29 If the reorganisation time of the environment is not muchfaster than the one of the system coherence is prolonged. However, it does notincrease the efficiency of energy transfer, except when excitation decay competeswith trapping.29 An experimental study on the same antenna protein was pre-sented at this meeting in a poster by Battacharyya and Sebastian. Mechanisms forthe dephasing of coherence by the environment were discussed in posters byKayal et al. and Roy et al., and the mechanism of photon-induced intra-molecularexcimer formation and charge transfer was treated by Mishra et al.30

In photosynthetic antennae the excitation decay does not to compete withtrapping. This is has been corroborated by the following recent experiments. VanGrondelle's group analysed the excitation transfer between six pigments (4 chlo-rophyll a, 2 pheophytin a) in the puried core of PSII.31 Quantum beats persistedfor several 100 femtoseconds even at ambient temperature. Several coherentexcimer-/charge-transfer states coexisted before the creation of a metastableradical pair. The authors suggested that quantum design of the core is pivotal forthe high quantum yield.31 In these experiments the core of PSII is isolated from itsantennae complement and it is excited by femtosecond pulses. In vivo the core issurrounded by antennae. Quanta trickle in at a rate of much less than 1000 persecond. Holzwarth and his colleagues studied this situation in oxygen evolvingPSII particles containing a complement of about 80 chlorophyll molecules.32,33 Toavoid singlet–singlet annihilation they kept the photon density of the excitingpulse low. The total time for exciton trapping and charge separation was about100 ps.32 The calculated intrinsic time of charge separation in the core, on theother hand, was much shorter, 2.7 ps.33 This situation is paralleled in purplephotosynthetic bacteria. Excitation transfer in the B850-ring of the antenna LH2as well as in the B875-ring of LH1 is very fast (about 100 fs) and it involves



Fig. 2 Structural model of the redox cofactors in the core of cyanobacterial PSII (modifiedwith permission of Science).114 The electron transfer uses the D1-branch of the hetero-dimer. Chorophyll a molecules are given in green and blue, pheophytin a in blue and theprimary (QA) and secondary (QB) quinones in purple. The catalytic CaMn4-cluster is linkedvia a tyrosine (TyrZ) to the chlorophyll (PD1). Centre-to-centre distances in A-units.


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coherence. The transfer from LH2 to LH1 takes 3 ps, and further on from LH1 intothe trap 35 ps.25 The long and trap-limited lifetime of excitation greatly exceedsthe decay time of coherence. Independent of whether coherent transfer isinvolved or not, the only condition for high quantum yield is that the transferbeats the dissipative loss of excitation in the whole system. This physicalconstraint is rather mild, and it provides a leeway for various transfer mecha-nisms and structural designs. In fact, photosynthetic organisms use a widepalette of mechanisms for excitation transfer, ranging from hopping by Forster-resonance over Frenkel-excitons to more delocalized modes involving thecoherent states of many pigment molecules. Accordingly, the antennae structurevaries considerably between organisms. Chlorophyll molecules are eitherenwrapped in proteins as in the LHC2 of plants,34 and in the LH1 and LH2 ofpurple bacteria,35,36 or self-organized as in the chlorosomes of green bacteria.37

The energy transfer may be directed towards a deep trap (low energy trap), as inPSI, or undirected if the trap is shallow, as in PSII.33 In all cases the quantum yieldof trapping is close to one.

4 Electron transfer

Excitation transfer into the very core of any reaction centre results in a very rapidcharge separation. The rst metastable product is a radical pair which is formedin picoseconds. In PSII this pair is pheophytinD1

+–chlorophyllD1� (see Fig. 2).

Herein the subscript D1 refers to the subunit to which the respective pigment isattached. While the positive charge is localized on this particular chlorophyll inPSII (PD1 in Fig. 2), it is delocalized over two bacterio-chlorophylls, the “special




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pair”, in the reaction centre of purple bacteria.38 The delocalization of theunpaired electron over this “special pair” hence is not pivotal for a high quantumyield, in contrast with previous beliefs.39

The charge separation is directed across the membrane. The membrane ischarged by 50 mV (eld strength 107 V m�1) per single turnover of each photo-system. Light induced voltage transients were rst detected in plant chloroplasts.Electrochromic absorption changes of intrinsic pigments served as a molecularvoltmeter40,41 with a very high time-resolution.42 The vibrational Stark-effect as amolecular voltmeter was discussed in a poster of this Faraday Discussion by S.Bagchi et al.

Structural pseudo-C2-symmetry is a common feature of most reaction centres.Their hetero-dimeric structure is considered an evolutionary legacy of a homo-dimeric and truly C2-symmetrical common ancestor.43–46 Anaerobic green bacteriastill host a homo-dimeric reaction centre.43

Independent of the type of reaction centre and organism, the positions andorientations of the six pigments in the core are very much the same. There arethree pigments on each branch of the dimer. While only one branch is redox-active in type II centres (of purple bacteria and in PSII of plants and cyanobac-teria), both are similarly active in a type I centre (PSI). The functional asymmetryin the former has been attributed (by Stark-effect spectroscopy) to differentdielectric screenings in the two branches.47 The asymmetric activity of type IIcentres is linked to its function as a one-electron to two-electron gate, whereasPSI, with its longer line of secondary acceptors, functions mono-electronically(for a thorough discussion and references see ref. 48).

The mechanism of electron transfer between cofactors that are embedded inthe protein is adequately described by the Marcus theory.49,50 It models tunnellingwithin the limits of the Born–Oppenheimer approximation. The rate is a functionof the (edge-to-edge) distance between donor and acceptor (r), and it depends onthree parameters: the standard free energy difference between the donor and theacceptor (DG0), the reorganization energy of their environment (l) and the decayconstant of wave-function-overlap (b):

rate � exp

"ðDG0 þ lÞ2

4lkT

#expð�brÞ (1)

This simple relation in terms of only three parameters has been experimentallyvery well met. In synthetic donor–spacer–acceptor triads it holds over three ordersof magnitude.51 As for the electron transfer between cofactors in a protein, variouspathways were screened in cytochrome c. Its heme served as the electron acceptorfor a photosensitizer which was covalently attached to various positions at thesurface. The transfer rate decreased depending on whether the electron passedmainly along single bonds, hydrogen bonds or through a void volume in theprotein.52,53 In a related study, Dutton and colleagues54 analysed the wealth ofkinetic and structural data on the electron transfer in photosynthesis and cellrespiration. They corrected the observed rates for the matching driving force(determined!) and reorganization energy (assumed!), namely �DG0 ¼ l. Over 13orders of magnitude the normalized rates decline mono-exponentially with theedge-to-edge distance (r) between donor and acceptor. Deviations by one order of




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magnitude can be attributed to local variations of l and b. The donor–acceptordistance is hence the major determinant of the rate of electron transfer.55 Thereorganization energy (l) and the decay constant of the electronic wave functions(b) in proteins do not vary too much. Nature's engineering of structures for rapidelectron transport has thus relied mainly on the distance between redox-cofactors(r) and on the driving force (DG0).55

Once the core of the reaction centre is hit by an excitation the primary electrontransfer is very fast. In PSII the radical pair (chlorophyll+–pheophytin�) appears inpicoseconds. The electron is then transferred to a rst quinone (QA) in about 400picoseconds and from there on to a second quinone (QB) in some 100 micro-seconds.56,57 The electron hole, on the other hand, is rapidly transferred in someten nanoseconds to a tyrosine58 and further in microseconds to the Mn4Ca-cluster. All of these reactions outrun by orders of magnitude the respectivewasteful back-reactions of excited states and radical pairs. Rapid forward reac-tions and slow back-reactions guarantee the high quantum yield of photosyn-thesis. On the other hand they require losses of free energy. In other words,photosynthesis sacrices energy efficiency for directionality.48

5 Water oxidation

PSII is a water–quinone oxidoreductase. The structure of cyanobacterial PSII isresolved at 3.8 A,13 3.5 A,59 and 1.9 A.14 Water (-derivatives) are oxidized by thecatalytic Mn4Ca-cluster and oxygen plus protons are liberated. When dark adap-ted PSII is excited by a series of short light ashes the Mn4Ca-cluster is clockedthrough sequentially higher and metastable oxidation states, until reaching thehighest state (see ref. 60 and references therein). Only then the reaction withbound water proceeds (in one millisecond) to yield dioxygen (see ref. 61). Thestepwise accumulation and pooling of four oxidizing equivalents, before initi-ating what still appears as the reaction of four-electrons-at-once, serves twopurposes. It homogenizes the energy demand of successive electron transfersteps, and controls hazardous intermediates (e.g. hydroxyl radical and super-oxide) on the way from water to dioxygen. There is a wealth of detailed kineticstudies on water oxidase, addressing the chlorophyll-moiety, an intermediatetyrosine, the Mn4Ca-cluster, electrons, protons and oxygen. A wide range ofoptical (X-ray, UV, VIS and IR), magnetic and mass spectroscopic techniques havebeen utilized to resolve electron transfer, proton transfer, conformationalchanges of the protein, substrate binding and product release in the time-domainfrom nano- to milliseconds (for reviews and references see ref. 62–65).

The precise structure of the manganese cluster is still under contention.During standard X-ray exposure of PSII-crystals Mn4 is the prime target of radi-ation damage66,67 (for radiation damage of metal markers for protein structuralstudies see Helliwell et al.).68 The reduction of Mnmay inuence its ligation state.The lack of unequivocal structural models of the Mn4Ca-cluster in its sequentialoxidation states is a major obstacle for a thorough understanding of the nalreaction cascade. For the time being, two non-invasive approaches, namely bymagnetic resonance spectroscopy (Endor69) and by theoretical chemistry (densityfunctional theory71), have converged towards one particular structural model ofthe CaMn4-cluster and its ligands including water (derivatives). X-ray crystalstructural analysis will eventually take up, either challenging or corroborating the




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present structural concept by a probe-before-destroy-approach (for principles andperspectives of this technique see Miller et al.).70 PSII-crystals have been exposedto an ultra-short and intense X-ray pulse (100 fs) of a free-electron-laser.72,73 Thefeasibility of analysing the cluster-structure in its sequential oxidation states hasbeen clearly demonstrated, although at a limited resolution so far (5–5.5 A).73

Structural details on the Mn4Ca-moiety, bound water (derivatives), and aminoacid ligands is the essential complement of kinetic data when aiming to under-stand the detailed reaction mechanism of this “holy grail” of photosynthesis.

6 ATP synthesis

Mitchell's daring hypothesis on proton-driven ATP synthesis7 roused the unpre-pared bioenergetics community. Soon thereaer essential elements of hishypothesis were detected in plant chloroplasts. Illumination generates trans-membrane voltage40 and a pH-difference,74 and the pH-difference75 and/or thevoltage76 can drive the synthesis of ATP. The molecular structure of ATP synthasewas then still unknown. Only its bipartite construction with a membrane portion,FO, which conducts protons, and a peripheral portion, F1, which interacts withnucleotides, was apparent. Boyer (Nobel Prize 1996) and his colleagues found thatF1 hosts two, if not three identical catalytic sites, that operate alternately,77 if notin a rotary mode.78 Conformational energy stored in the enzyme might drive theformation of ATP from ADP and P1.79 In 1994 Walker (Nobel Prize 1996) and hiscolleagues presented the rst structural model of its chemical generator at a 2.3 Aresolution,15 now at 1.9 A,80 which strongly favoured a rotary function of F1. F1 is apseudo-hexagon of two types of subunits arranged as (ab)3 with a cranked sha(subunit g) in its centre. It suggests that the rotation of the central sha drivesATP synthesis when progressing from one catalytic site on b to the next. Soonthereaer the rotation of the central sha was experimentally established, start-ing without time resolution by the biochemical crosslink technique,81 and thentime resolved either by polarized photobleaching and recovery82,63 or uorescencemicroscopy.83 The latter technique became the gold standard in the eld(for original video recordings see ref. 119). Its principle is illustrated in Fig. 3a.The hexagonal body of the enzyme is xed on a solid support so that the hydro-lysis of ATP drives the sha around. In a pioneering experiment, a uorescentactin lament was attached to the foot of the central stalk of F1 and the rotation ofthe lament was video-recorded in a uorescence microscope.83 In the author'slab the uorescent probe was attached to the holo-enzyme, FOF1, as shown inFig. 3a. Notably the procession of this enzyme over its reaction coordinate can berecorded in real-time. By certain tricks it has been feasible to correlate thedynamic jumps and transient dwells of the live enzyme with the still pictures ofthe inhibited enzyme as a crystal structure.84–86 Moreover, the rotor position hasbeen externally manipulated by an attached hyper-paramagnetic bead.87,88

Ongoing studies in this line will likely provide more precise data on the energylandscape of F1, a prerequisite for thorough theoretical descriptions (see ref.89–91 and references therein). The wealth of structural and kinetic data qualiesATP synthase as a hydrogen atom of nano-motoring. The sophisticated, yet simpleelectro-mechano-chemical operation of ATP synthase has been described inseveral reviews.6,17–20,92,93 For animations of the enzyme and its two motors, FO andF1, see ref. 121. A few salient features shall be emphasized in the following.



Fig. 3 The micro-videographic assay for the rotary activity of FOF1.115,116 (a) The holo-enzyme is solubilized in detergent and the crown of F1 is attached by His-tags to a solidsupport. A fluorescent actin filament is attached to the rotor ring of FO (modified withpermission from FEBS Lett.116). (b) When ATP hydrolysis drives the rotor ring round, theactin filament, subject to viscous drag on the filament, is curved. The elastic parameters ofthe filament are determined by fluctuation analysis (modified figures from ref. 115 withpermission). For the original video-recordings see ref. 120.


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Both FO and F1 are stepping motors. When driven by ATP hydrolysis F1 rotateswith a period of 120�, with substeps of 40� and 80�.94–96 This stepping reects thepresence of three equivalent reaction sites on F1 and torque production duringsubstrate binding, catalysis and product release. On the other hand FO steps by36�,97,98 reecting the C10 symmetry of the particular bacterial FO. The mechanismand the structure of the rotary ion-motor, FO, is not discussed here. For theprinciple of operation see ref. 18, for the kinetic properties ref. 99, for thestructure ref. 16 and for simulations of its reaction dynamics ref. 89,99,100. Herewe focus on the cooperation of FO and F1 in the holo-enzyme.

If the proton-motive force is larger than the thermodynamic force of ATPhydrolysis the electrochemical motor of ATP synthase, FO, drives the chemicalgenerator, and F1 synthesizes ATP. If the force-relation is reversed, motor andgenerator change roles. ATP hydrolysis by F1 drives FO to generate the proton-motive force. If the forces match the enzyme rests. Thermodynamic quasi-equi-librium between the two motors was used to determine the torque generated byATP hydrolysis.101 As illustrated in Fig. 3a a long lament (�3 mm) was attached tothe rotary electromotor of the holo-enzyme. It slows the turnover rate of theenzyme by several orders of magnitude into a state of quasi-equilibrium. Thetorque was calculated from the elastic deformation of the lament (documentedin Fig. 3b), which reects the counteraction of the drive and the viscous drag. Themean torque, 56 pN nm, conformed with the calculated driving force ofATP-hydrolysis (70 kJ mol�1),101 which implies that the two motors do not slipagainst each other.

Although torque production by F1 was expected to progress in steps (under thegiven conditions), the torque output to the actin lament on FO was almostconstant. This surprising observation has been explained by solving the Fokker–Planck equation of a stepping nanomotor when elastically coupled to a heavy




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load.101 Decreasing the stiffness of the elastic buffer between the stepping driveand the load attens the torque prole at the output. It increases the turnover rateby orders of magnitude over the one with a stiff transmission (see Fig. 4a). Inother words, an elastic force–torque transmission is pivotal for high kinetic effi-ciency of the stepping nanomotor that drives a heavy load.102 This benet of acompliant transmission applies to all of nature's intrinsically stepping nano-motors. The distribution of elastically compliant and stiff domains over FOF1 wasdetermined by uctuation analysis.20,103,104 The result is illustrated in Fig. 4b. Themost compliant domain (stiffness 70 pN nm) of the holo-enzyme is located on thecentral rotor and between the sites where the respective power strokes aregenerated (see ref. 6 and 20 for details and references). The rest of the enzyme,and in particular the stator, are stiffer by an order of magnitude. The elastictransmission decouples the two motors kinetically while keeping them strictlycoupled, both thermodynamically and, in the time average, also under a steadyturnover. It explains why this enzyme can run with different gears in differentorganisms. The gear ratio of FO : F1, and likewise the expected H+ : ATP ratio,ranges from 8 : 3 in mammalian mitochondria105 to 10 : 3 in yeast mitochon-dria105 and 14 : 3 in chloroplasts,106 and it varies between 10 : 3 and 15 : 3 indifferent bacteria (see ref. 107 and references therein). Organisms (organelles)thriving at a large and constant ion-motive force, like mammalian mitochondria,run at low gear (speedsters), and those at a low and/or uctuating force, likechloroplasts and in alkaliphilic bacteria, at high gear (tractors).6 Because the twomotors are kinetically decoupled the enzyme can operate with a strictly protonspecic ion motor in some organisms,99 and optionally on sodium or proton108,109

Fig. 4 On the elastic torque transmission between the ion-driven motor, FO, and thechemical generator, F1, of rotary ATP synthase. (a) The distribution of elastically compliant(red) and stiff domains (grey) over the enzyme. Numbers give the torsion stiffness in pNnm.6,117,118 (b) Calculated dependence of the turnover rate of a stepping and rotarynanomotor, which is coupled to a heavy load, on the torsion stiffness of the transmissionto the load.115 With permission from the Annual Review of Biochemistry, Volume 84. ©2015, Annual Reviews, http://www.annualreviews.org.




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in others, depending on the ambient ion concentration. The elastically complianttransmission accounts for considerable freedom of design for both rotor andstator in the similarly bipartite relatives of FOF1, namely the archeal A-ATPase andthe V-ATPase of eukaryotes.6,110 They all share with other nucleotide triphospha-tases the (pseudo-)hexagonal design of the catalytic headpiece, namely with hel-icases and bacterial DNA-translocases which rotate and/or translocate proteins,RNA or DNA, in their central cavity and probably share a common ancestry.111

7 Outlook

Oxygenic photosynthesis in its present and fossil forms provides man with food,ber and fuel. The quantum yield of photosynthesis is almost perfect. As toprimary energy efficiency in relation to the solar spectrum (about 20%), oxygenicphotosynthesis compares well with single-band-gap photovoltaic cells.112 For theyearly average of crops in the eld however the efficiency drops down to 2%, andfor bio-ethanol production to 0.2%, if not being energy negative (as were biofuelsof the rst generation). The total useful energy of biomass which is globallyproduced in one year (on land) is only about 5-times greater than man's yearlyenergy consumption. It is obvious that present day biomass, and even more sobiofuels, cannot satisfy man's ever growing energy demand. Although bio-ethanolis produced and used on a large scale in Brazil, it is no option for countries withhigher population densities and/or industrialization, namely China, the EU andIndia. The products of photosynthesis should thus rather be reserved for food,feed, bre and platform chemicals. A sustainable energy supply calls for technicalutilization of solar energy. Wind-power, photo-thermal and photo-voltaic devicesare technically established, economically competitive, and increasingly installedworldwide. It is a major scientic challenge to develop new techniques for theproduction of solar fuels, articial photosynthesis with greater efficiency, andbased on low cost catalysts.113

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119 Original video recordings of rotarymotion of F1: http://www.k2.phys.waseda.ac.jp/Movies.html.

120 Original video recordings of FOF1: http://www.home.uni-osnabrueck.de/wjunge/Media.html.

121 Animations of the rotary activity of F1, FO, and FOF1: http://www.mrc-mbu.cam.ac.uk/research/atp-synthase/molecular-animations-atp-synthasehttp://www.home.uni-osnabrueck.de/wjunge/Media.html.



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