J. R. Soc. Interface pursuit and characterization of hydrogen tunnelling in doi:10.1098/rsif.2006.0122 a variety of oxidoreductases. Fifteen years later, Published online Chance first revealed that quantum mechanical electron tunnelling was a critical part of biological oxidation and reduction using light-active photosyn- thetic bacteria at cryogenic temperatures. In the classic REPORT 1966 paper with the late Don DeVault, he not only demonstrated the tunnelling signature of temperature insensitive rate constants between 77 and 5 K for the Quantum catalysis in millisecond oxidation of cytochrome c by the light- activated bacteriochlorophyll but also provided a enzymes: beyond the theoretical basis for a long-distance electron tunnelling of over 20˚ A. Fifty years ago, Rudolph Marcus transition state theory (Caltech) first penned his classical theory of oxida- tion–reduction reactions (equation 1.1) involving paradigm. A Discussion electron transfer in solution. In the 1970s, these Meeting held at the Royal equations became the foundation of experimental 1 KðDG ClÞ2 Society on 14 and k f pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi exp ð1:1Þ et 4lk T 15 November 2005 4plkBT B investigations into biological electron tunnelling, again 1, 2, using photosynthetic systems. The early 1980s saw the Nigel S. Scrutton *, Michael J. Sutcliffe * first heroic experimental demonstration by John Miller and P. Leslie Dutton3,* and colleagues of the parabolic rise of the log rate constant with increasing driving force (KDG)toa 1Faculty of Life Sciences, and 2School of Chemical maximum (defining the reorganization energy, l) Engineering and Analytical Science, University of followed by a fall in the ‘inverted region’ as predicted Manchester, Jackson’s Mill, Manchester M60 1QD, UK by Marcus. The relatively shallow fall of the inverted 3Department of Biochemistry and Biophysics, region was indicative of strong coupling of the electron University of Pennsylvania, Philadelphia, tunnelling to quantized vibrational modes and hence the PA 19104-6059, USA tunnelling of nuclei as well. Application of Marcus theory has become one of the foundations of transition How do enzymes work? What is the physical basis of state theory and a mainstay of those working on the the phenomenal rate enhancements achieved by Discussion Meeting’s principal subject, tunnelling of the enzymes? Do we have a theoretical framework that hydrogen nucleus in enzymatic oxidation and reduction. accounts for observed catalytic rates? These are the Chance opened the meeting with an outline of the foremost questions—with particular emphasis on early developments by Keilin and Warburg in respir- tunnelling phenomena—debated at this Discussion ation and of Hill in photosynthesis that paved the way Meeting by the leading practitioners in the field. to the discovery of tunnelling in biological electron transfer reactions. He emphasized the early importance Keywords: quantum tunnelling; electron transfer; of purple photosynthetic bacteria in facilitating the hydrogen transfer; transition state theory; demonstration of electron tunnelling, and the later enzyme catalysis importance of the highly robust photosynthetic reaction centre proteins that offered an unprecedented breadth of biochemical and biophysical experimental scope to explore the parameters of Marcus theory in 1. BIOLOGICAL ELECTRON TUNNELLING biology. Les Dutton (University of Pennsylvania, USA) introduced the progression of work that put to test the This Discussion Meeting was fortunate to have the parameters in the quantized form after Hopfield presence of two distinguished colleagues who have (equation 1.2a and 1.2b). In the 1970s and played critical roles in the foundations and develop- ment of tunnelling mechanisms in biology, Britton K C 2 f ffiffiffiffiffiffiffiffiffiffi1 ðDG lÞ Chance (University of Pennsylvania, USA) and ket p exp 2 ; ð1:2aÞ 2ps2 2s Rudolph Marcus (California Institute of Technology, Zu USA), both Foreign Members of the Royal Society. s2 Z lZu coth ð1:2bÞ Moreover, 70 years ago Chance invented the stopped- 2kBT flow spectroscopic instrumentation that led to the proof 1980s, his laboratory’s work on reaction centres made of the Michaelis enzyme–substrate complex. This immediate use of technical opportunities such as ultra- complex was very much the focus of attention during fast lasers for activation for tunnelling kinetics over a the meeting that discussed theory, experimental 300 K range (rate constants and Zu) and replacement of cofactors and mutagenesis to change the driving *Authors for correspondence ([email protected]; force by as much as an electron volt and estimate [email protected]; [email protected]). reorganization energies (DG and l). Reaction centres

Received 3 March 2006 Accepted 3 March 2006 q 2006 The Royal Society 2 Quantum catalysis in enzymes N. S. Scrutton and others were the first membrane proteins to yield high- hydrogen, and barriers to the reaction that reflect resolution crystal structures that provided critical environmental reorganization, was notable at the inter-cofactor edge-to-edge distances (R) and atomic meeting. This is a significant departure from views level details of protein packing (r). Dutton reported on held until recently by many in the field that tunnelling how these Marcusian tunnelling parameters are is restricted to a few enzyme systems, and where selected and varied in biology in the natural engineering observed, can be interpreted using the Bell ‘tunnelling of oxidoreductases. This Darwinian examination at the correction’ of semi-classical transition state theory. molecular scale yields a remarkable simplifying picture. Kohen presented his experimental studies of H-tunnelling One by one, the parameters were shown to be more or in dihydrofolate reductase. He emphasized the role of less generic components of the engineering (Zu, r and l distal mutations on enzymic H-tunnelling probed varying but not selected), leaving a clear and dominant through measurement of kinetic isotope effects and selection of distance for the engineering of electron their temperature dependence. Using isotopically transfer chains and catalytic clusters. DG is sometimes labelled cofactors, he presented kinetic data that are selected and sometimes not, especially when the consistent with full tunnelling models for the hydrogen distance between the redox cofactors is small. It is nucleus. His kinetic studies with mutant enzymes also not unusual that electron tunnelling through altered at residues distal to the active site—residues chains encounters major endergonic redox step(s) (up that are predicted theoretically to be part of a network to several hundreds of meV) in electron transfer chains of motions coupled to the active site—provide exper- that overall are mildly exergonic. Dutton provided test imental support for coupling of motion to active site drives for the resultant Darwin-purged quantized chemistry, and are consistent with environmentally Marcus expression (equations 1.3a and 1.3b) coupled models of H-transfer. Dihydrofolate reductase C 2 featured also in the presentation of Ruedi Allemann exer ðDG lÞ log ket Z 15K0:6RK3:1 ð1:3aÞ (University of Cardiff, UK). Unlike Kohen’s approach l which relies on measurements in the steady-state and K C 2 ender ð DG lÞ DG analysis of the commitment to catalysis to extract log k Z 15K0:6RK3:1 K ð1:3bÞ et l 0:06 intrinsic kinetic isotope effects, Allemann ‘isolated’ the in several simulations of native electron transfer chemical step using stopped-flow methods. He empha- systems. This included (i) light-activated electron sized studies with a thermostable dihydrofolate tunnelling mediated charge separation and recombina- reductase from the hyperthermophile Thermotoga tion in the highly complex electron transfer chains of maritima as well as the intensively studied Escherichia the reaction centres of plant photosystems; (ii) electron coli enzyme and he presented kinetic data consistent transfer through the long chain of nitrate reductase with environmentally coupled models of H-tunnelling. that includes a 600 meV thermal redox barrier that Of interest was his finding that at physiological pH, the marshals multiple electrons before delivery to the kinetic isotope effect data are consistent with the need catalytic site and (iii) the role of the barriers of proton to invoke ‘gated motion’ along the H-transfer coordi- pumping and dioxygen reduction in cytochrome oxi- nate, but at elevated pH the reaction was dominated by dase in removing the possibility of electrons by-passing passive motions. the catalytic site. Dutton also noted the power of Judith Klinman (University of California–Berkeley, shortened electron tunnelling distances between cofac- USA) developed further the theme of environmentally tors at the thresholds of catalytic sites. He identified the coupled hydrogen tunnelling by reference to her work energetic limit in two-electron catalysis for two, with soybean lipoxygenase. She emphasized recent sequential one-electron tunnelling to a thermally studies with wild-type and mutant enzymes that accessible radical transition state. Beyond this limit, exhibit very large primary deuterium kinetic isotope when the one-electron reduction is energetically effects and the notion of ‘distance sampling’ required to remote, a dramatic mechanistic change must occur bring donor and acceptor atoms sufficiently close to towards the principal subject matter of the meeting, the effect efficient wavefunction overlap. The wild-type concerted two-electron transfer and tunnelling as a enzyme has a very small enthalpy of activation, and hydrogen or hydride. that for deuterium transfer is only slightly increased. The primary kinetic isotope effect is therefore weakly dependent on temperature and the pre-exponential factor ratio (A /A ) derived from Arrhenius analysis is 2. ENZYMIC HYDROGEN TUNNELLING H D substantially inflated over that expected for semi- Measurement of competitive kinetic isotope effects and classical transfer. Klinman interprets these data to their temperature dependence is now the ‘gold stan- indicate an optimized active site set up to catalyse dard’ for diagnosing experimentally tunnelling regimes H-transfer by tunnelling. The temperature dependence in enzyme systems. Amnon Kohen (University of Iowa, of the reaction is increased in mutant enzymes altered USA) provided an insightful review of these on either side of the donor carbon atom of the substrate. approaches, now widely adopted in the field, and The size of the kinetic isotope effect remains constant, discussed recent data from a number of laboratories, but the AH/AD prefactor ratio is reduced substantially including his own, that are consistent with environ- below unity. These data are interpreted to indicate mentally coupled or vibrationally assisted tunnelling a less optimal configuration for tunnelling in the models. The more general acceptance of full tunnelling mutant enzymes, leading to increased activation models that accommodates tunnelling of all isotopes of energy. The Klinman analysis is based on, and fully

J. R. Soc. Interface Quantum catalysis in enzymes N. S. Scrutton and others 3 consistent with, the full tunnelling model for enzymic containing orthogonal electron transfer and proton H-tunnelling and, coupled with structural analysis of transfer pathways—thereby allowing the electron and lipoxygenase offers new insight into the effects of proton tunnelling events to be disentangled—were localized mutations on tunnelling characteristics. presented. The lessons learnt from these model Experimental studies of tunnelling in B12-dependent systems enabled PCET to be exploited in biomimetic systems, specifically methylmalonyl–CoA mutase, were mono-oxygenases—utilizing the ‘Hangman’ porphyrin also presented by Ruma Banerjee (University of architecture—which displayed unprecedented multi- Nebraska, USA). In this system, the transfer of a functional catalytic activity. The flash activation hydrogen atom from substrate to the deoxyadenosyl method developed on the model systems was used to radical is accompanied by a very large kinetic isotope resolve the proton coupling in an enzyme system—the effect, a value that is well beyond the upper limit for radical-based quantum catalysis in class I E. coli semi-classical reactions in the absence of tunnelling. ribonucleotide reductase. This enzyme utilizes both The reaction is kinetically complex involving cobalt– co-linear and orthogonal PCET to transport charge carbon bond cleavage, a homolysis reaction that is from an assembled diiron–tyrosyl radical cofactor to ‘triggered’ by conformational change in the protein. the active site over 35 A˚ away via an amino acid radical Banerjee presented an elegant computational analysis hopping pathway spanning two protein subunits. of the cobalt–carbon bond cleavage reaction which Ma˚rten Wikstrom (University of Helsinki, Finland) provided details of the energetics of formation of the presented a study of PCET in cytochrome oxidase. radical products of homolysis. The simulation demon- Recently, it has been suggested the proton pumping in strated the ideal geometry of the radical species for H cytochrome oxidase is not mechanistically coupled to atom transfer from substrate to the deoxyadenosyl electron transfer, but instead to proton consumption at radical following cobalt–carbon bond cleavage. Dexter the active O2 reduction site. Time-resolved optical Northrop (University of Wisconsin-Madison, USA) spectroscopy was used to address this, showing that presented interesting data on the pressure dependence electron transfer from the low-spin haem a to the active of isotope effects in enzymes and made a case for O2 reduction site is in fact kinetically coupled to the hydrogen not tunnelling in yeast alcohol dehydrogen- primary event of the proton pumping mechanism—an ase. His work has demonstrated suppression of kinetic internal proton transfer from a conserved glutamate to isotope effects at high hydrostatic pressure in this an as yet unidentified site above the haem groups. enzyme system, which finds similarities with studies of chemical reactions by Isaacs that display unusually large kinetic isotope effects. Isaacs had previously 3. THEORETICAL CONSIDERATIONS AND shown that ‘normal’ deuterium kinetic isotope effects COMPUTATIONAL SIMULATION are insensitive to pressure, but those reactions display- Further detailed insight, particularly at the atomic ing an unusually high kinetic isotope effect reflecting level, comes from theory and simulation—such studies inferred contributions from quantum mechanical tun- are key to corroborating and extending findings that nelling are sensitive. Northrop originally attributed his emerge from experimental studies. Sharon Hammes- pressure sensitivity of the kinetic isotope effect Schiffer (Pennsylvania State University, USA) gave a measured with alcohol dehydrogenase to tunnelling clear exposition of how theory and simulation can give effects, but he now rules out tunnelling effects on the valuable insight into the impact of enzyme motion on basis of more recent data and extrapolations of the enzymic hydride and PCET. Studies of PCET were intrinsic kinetic isotope effect to unity at pressures illustrated for the reaction catalysed by lipoxygenase, above the experimental range. Moreover, 13C isotope using a formulism that treats the active electrons and effects are also suppressed at high pressure, which he transferring protons quantum mechanically. These uses to argue against significant tunnelling effects in the showed that the small overlap of the reactant and H-transfer reaction and he offers protein domain motion product proton vibrational wavefunctions gives rise to as a possible origin of the observed effects. Pressure the highly elevated experimentally determined proto- effects are delivering new types of data that are n/deuterium kinetic isotope effect. The study illus- seemingly challenging current frameworks for the trated that proton donor–acceptor vibrational motion physical basis of H-transfer. The mechanistic basis of plays a vital role in PCET. The role of motion in the these effects generated is as yet unclear, but generated hydride transfer reaction catalysed by dihydrofolate much debate at the meeting. It is clear that studies of reductase was also investigated. The hybrid quantum/ this type will make a major contribution to current classical molecular dynamics simulations—using the experimental strategies based on more established empirical valence bond approach—showed that nuclear methods, i.e. competitive and temperature-dependent quantum effects such as zero point energy and hydrogen isotope effects studies. tunnelling are significant in these reactions, where they Dan Nocera (Massachusetts Institute of Technology, lower the free energy barrier significantly. The simu- USA) presented experimental studies of proton-coupled lations also identified a network of coupled motions electron transfer (PCET)—intrinsically a quantum extending throughout the enzyme—these correspond to mechanical effect since both the electron and proton conformational changes along the collective reaction tunnel. Nature has resolved the fundamental difference coordinate. Importantly, these coupled motions facilitate in transfer distances by the evolution of enzymes to hydride transfer by reorganizing the environment— control proton transfer (shorter) and electron transfer decreasing the donor–acceptor distance, orienting the (longer) on very different length scales. Model systems substrate and cofactor appropriately, and providing

J. R. Soc. Interface 4 Quantum catalysis in enzymes N. S. Scrutton and others a favourable electrostatic environment for hydride USA) presented results illustrating that transition transfer. It was also shown that mutations distal to state theory could not fully describe the dynamics of the active site cause non-local structural changes that enzyme-catalysed reactions. Using a model system impact significantly on the probability of sampling comprising a double well coupled to an environment configurations conducive to hydride transfer, thereby and to a symmetrically coupled promoting vibration, it altering the free energy barrier and the rate of hydride was shown that reactive trajectories pass through a transfer. wide range of points, most of which are not the saddle The role of protein motion in promoting tunnelling point. A study of lactate dehydrogenase addressed the also featured in the presentation by Mike Sutcliffe question of how motions, such as promoting vibrations, (University of Manchester, UK). He amply demon- which are on the picosecond time-scale can be strated the power of experiment (Nigel Scrutton, reconciled with the millisecond time-scale of enzymatic University of Manchester, UK) and theory pursued turnover. The study illustrates the answer lies in the in close partnership in the same laboratory. His large conformational space that has to be searched to simulations of proton tunnelling steps—based on find the rare conformation needed to produce the atomic resolution structures of reaction intermediates required dynamic effects—only a small minority of and a variational transition state theory approach—- conformations appear to be catalytically competent. confirmed that tunnelling dominates in these Jose´ Onuchic’s (University of Southern California at reactions. Good agreement with experiment was San Diego, USA) presentation illustrated how recent obtained for both calculated kinetic isotope effects theoretical advances have shed light on the impact of and calculated phenomological activation energies protein dynamics on electron tunnelling and biochemi- when the contribution of tunnelling is included. cal reactivity. The original Pathway model has been Protons were shown to tunnel over relatively short generalized to understand how protein dynamics distances. Sutcliffe also discussed the role of long- modulate not only the Franck–Condon Factor but range coupled motions and shortrange motions in also the tunnelling matrix element. Dynamical effects promoting tunnelling through modulation of proton- are of critical importance when interference—particu- acceptor distance. Tunnelling in the related enzyme, larly destructive interference—among pathways modu- methylamine dehydrogenase, was presented by Neil lates the electron tunnelling interactions in proteins. Burton (University of Manchester, UK). With meth- Such dynamical modulation of the electron transfer ylamine as substrate, different tunnelling paths were rate has been able to explain and/or predict a number investigated across an ensemble of reactive enzyme of experimental rates that were subsequently confirmed configurations. The results are suggestive of two by experiment. Results were also presented that pathways, one of which is dominated by tunnelling illustrated how global transformations, which control and the other dominated by a classical ‘over-the- protein functions such as allostery, may involve large- barrier’ mechanism. The influence of a number of scale motion and possibly partial unfolding during the active site amino acids on proton tunnelling and the reaction event. utility of enzyme mimics were also discussed. Hans Limbach (Freie Universita¨t Berlin, Germany) pre- sented results illustrating the application of the one- 4. UNANSWERED QUESTIONS dimensional Bell–Limbach tunnelling model to several The summing up of, and presenting the future different hydrogen transfer reactions in small molecule perspectives for, such a lively and stimulating Discus- systems for which experimental kinetic data are sion Meeting requires someone with both a broad and available, calculating the temperature dependencies deep knowledge of the topics covered. Rudolph Marcus of rates and kinetic isotope effects. This study suggests (Caltech; Nobel Laureate and Foreign Member of the that analysis of Arrhenius curves using the Bell–Lim- Royal Society)—having played a critical role in laying bach tunnelling model is a useful approach for a first the foundations of and subsequently developing our screening of experimental data prior to more advanced conceptual framework for understanding tunnelling physical descriptions and theoretical interpretations. mechanisms in biology—very ably and wisely fulfilled In contrast to the results presented by the speakers this role. He perceptively identified that an important above—reflecting current controversy in the field— component of the continued successful advancement of Arieh Warshel (University of Southern California at the field is the need to develop a common language, e.g. San Diego, USA) presented results that challenge not when referring to dynamical effects. We need to agree only a role for dynamical effects in enzymic reactions, how we define transition state theory—is extra but also the very existence of tunnelling in such dynamics required in such a formulism and how reactions. Simulation of the lipoxygenase reaction important is re-crossing the barrier? He also pointed reproduces the very large observed kinetic isotope out that the introduction of new experimental techni- effect and the observed rate constant without quantum ques—for instance the use of pressure effects—heralds mechanical tunnelling. The results suggest that tran- the need for a generally accepted working framework to sition state theory provides a quantitative framework accommodate, explain and advance them experimentally for studies of enzyme catalysis, and that the key and theoretically. It was evident from Marcus’ sum- questions remaining to be addressed relate to the mary that further advancement into the realm of nature of the factors that lead to transition state hydrogen tunnelling is nevertheless still wrestling stabilization. However, in contrast Steve Schwartz with long-standing but non-trivial theoretical (Albert Einstein College of Medicine, New York, and experimental obstacles toward mechanistic

J. R. Soc. Interface Quantum catalysis in enzymes N. S. Scrutton and others 5 understanding. These include questions as basic as how theory paradigm of enzyme catalysis, and in bringing enzymes achieve catalysis and, in this regard, how many—including some new—key questions to the fore. important is it for reactant and product wells to be Very evident in the lectures and vigorous discussions symmetric for tunnelling to occur and, not least, how was the message that future progress and insights into can the effects of the common mutagenesis approach be this challenging interface of chemistry, physics and interpreted and used without ambiguity. This meeting biology can benefit from the synergy evident in a close served the broad biochemistry and biophysics fields partnership of experimental techniques and simulation well in presenting the view beyond the transition state and theory.

J. R. Soc. Interface