Photosynthesis Research 22: 5-10, 1989. © 1989 Kluwer Academic Publishers. Printed in the Netherlands.

Personal perspectives

Tunneling enters biology Don DeVault

University of Illinois, Department of Physiology and Biophysics 407 South Goodwin Avenue, 524 Burrill Hall, Urbana, IL 61801, U.S.A. Received 14 March 1989

Key words:

cytochrome, electron transfer, laser, low temperature, photosynthesis, quantum mechanics

I have been asked to write something about the experiment with Britton Chance which gave evidence for quantum mechanical "tunneling" in a biological system. The system was bacterial photosynthesis in Chromatium vinosum. The reaction was cytochrome photo-oxidation which is simply the transfer of an electron from a cytochrome to the photo-oxidized bacteriochlorophyll special pair of the reaction center. In 1960 Mitsuo Nishimura and Britton Chance (Chance and Nishimura 1960) discovered that the reaction occurs readily at 77 K. Brit recognized this as a rather unique low-temperature chemical reaction worthy of further study. Walter Bonner, also at the University of Pennsylvania, found low temperature cytochrome oxidation in green leaves and Brit joined him to study it (Chance and Bonner 1963). Brit had also recognized early the possibilities of using lasers to activate photochemical reactions and had built a ruby laser with Victor Legallais and published an experiment on photosynthesis with Heinz Schleyer (Chance et al. 1963). When I arrived at the Johnson Foundation late in 1963 he put me to work on a newly purchased ruby laser to pursue the C. vinosum kinetics. I had arrived from 20 years of "horsing around" in non-scientific endeavors. I got my PhD in 1940 at the U. of Cal. at Berkeley studying artificial radioactivity under Willard Libby in the chemistry department. In the same year I registered for the draft as a conscientious objector. After a couple of years of post-doctoral work under Philip A. Leighton at Stanford University I inconsistently went to work with Felix Bloch and colleagues in the physics department on a branch of the atomic

bomb project. (Bloch was persuasive and the temptation to work with such a great physicist was just too much, so I agreed tentatively.) I soon realized that it was wrong for me and after six months, at the end of 1942, I resigned. Early in 1943 Selective Service classified me for noncombatant service but I refused and got an 18 months jail sentence, the last half of which I served on parole in a so-called Civilian Public Service Camp for conscientious objectors. (I figured I deserved the sentence if for other reasons than the government's.) I also got a second sentence (31 years) for setting up a bacteriological laboratory in the barracks and experimenting with the physical chemistry of the cup assay method for antibiotics (penicillin was in the news at that time) instead of building ponds for wild ducks. I was paroled in the middle of 1946 and went to work under Bill Libby again, this time at the Institute for Nuclear Studies (INS) at the University o f Chicago. On my way to Chicago from the Federal Penitentiary at Springfield, Mo., I stopped off at St. Louis to have lunch with Martin Kamen whom I had known in Berkeley. Martin was having his own adventures with the government (Kamen 1986) and as he expressed it, we wondered which o f us was doing the other the most harm by being seen together. At Chicago I had a hard time concentrating on the science and spent most of my time with the Chicago chapter of CORE (Congress of Racial Equality) using Gandhian non-violent methods to fight racial discrimination. I recently wrote a small book, "Science and Satyagraha" (DeVault 1987) as a sort of report o f this research.

Fig. 1. The apparatus as of 1967. Going from left to right we have on the table at the extreme left the Q-switched ruby laser. Below it the laser power supply. Just below the table top, to the left of middle, the control box is screwed to the front of the table. Just above the control box would be the oscilloscope and camera, absent when the picture was taken. Also absent is the aluminum tube in front of the oscilloscope thru which the laser beam passed and which protected the beam from interference by hands. Below the control box is the circuitry for the lamp-boost etc. and the power supply for the photomultiplier. To the right of middle on the table is the laser attenuator box (concave lenses inside). At the right is the liquid helium cryostat. It is loaded at the top, with the help of a ladder, and the sample chamber is at the bottom. The monochromator for the measuring light is behind the cryostat. The vacuum pumps are out of the picture. The parts on the table are mounted on several separate boards resting on sponge rubber pads to damp the vibrations from the spinning prism of the laser. (From Chance et al. (1967) with permission of the present copyright owners, Norstedts Forlag, Stockholm)

I met my wife in 1948 and quit the INS to seek an "intentional community" in which to live, a little ahead of the "hippie" movement. We lived 10 years in Tracy, Calif., during most of which I taught physics and physical chemistry at College (now University) of the Pacific. We and our two children then spent a year at Koinonia Farm near Americus, Georgia, and then moved to Philadelphia and vicinity. Activities there, before joining the University of Pennsylvania, included working for a small electronics company learning transistor circuitry while helping to develop a guidance device for blind people. We protested the building of the Kinzua Dam on the Allegheny river which would flood much of the Seneca Indian reservation and also joined the protest against preparation for germ warfare at Ft. Detrick in Maryland. That is background. When I arrived at the Johnson Foundation I had

been out of science so long that I didn't even know what " A T P " was. My total background in photosynthesis was an acquaintance with the Ruben, Kamen, Hassid group across the hall from me when I was a graduate student. Martin Kamen has written about this exciting work in several places (Kamen 1974, 1986). They were tracing carbon with the C-11 isotope (20min half-life). They put my name on one of their papers (Ruben et al. 1939) I guess because I had joined in their "bull-sessions" where we tried to figure out how chlorophyll could chemically react with the CO2. Before the use of the laser all measurements of the rate of cytochrome photo-oxidation had been light limited. Brit's idea was that with a Q-switched laser we could saturate the photosynthetic system in a time so short that the true rate of oxidation could be measured immediately after absorption of the light. My first measurements at room tempera-

ture gave a halftime of 18#sl which I telegraphed to Brit attending a conference in Berlin (Chance and DeVault, 1964). Later I began to get measurements in the neighborhood of 2 #s which puzzled us for some time. The truth dawned when I realized finally that the advent of the 2 ps results coincided with my introduction of a new 2-transistor preamplifier that I had designed to replace the old cathode follower. (For those not familiar with the technique, the pre-amplifier couples the out-put of the measuring light detector to the oscilloscope or other read-out device.) Still faster amplifiers, pioneered by William Parson later brought the measurements of room temperature half-time down to 1 #s. We rigged up a system for cooling the cuvette with a stream of cold nitrogen gas from liquid nitrogen and measured the rate of cytochrome photooxidation at various temperatures down to 77K, where the half-time was about 2 ms. There was a hint that the rate had become constant below about 100K. Brit then bought a low temperature cryostat, an Air Products hydrogen liquefier. With help from Mr. Bruno Graf we extended the measurements to 30K and confirmed that the rate was constant below 100K. This resulted in our much mentioned 1966 paper, (DeVault and Chance, 1966). We then bought a liquid helium cryostat and, with John H. Parkes doing most of the work, found that the half-time, is constant at 2.3 ms from 100K to 4K (DeVault et al. 1967). We used dark-adapted anaerobic whole cells of C. vinosum insuring that it was the low potential cytochrome whose oxidation we observed. They were furnished to us by Jane Gibson, Heinz Schleyer and Margaret Edwards (Weiss). Bacteriochlorophyll (Bchl) assays were done by Mrs. Virginia Montgomery and Mr. Jeffrey Cohlberg who rendered much help. Also assisting were Mr. Ben Wu, Mr. James Cain and Dr. William Hildreth. The measurements are difficult because at lowtemperature the oxidized cytochrome does not recover and a new sample is required for each laser shot and also because one cannot expose the sample to much measuring light before the shot to adjust the signal offset required to keep the signal on the oscilloscope screen. The measuring-light itself could oxidize the cytochrome. Also in 1964 to 1967 we did not have signal digitizers or even

storage oscilloscopes fast enough for the measurements contemplated. Read-out was, therefore, by camera trained on the scope, photographing it live. The fast measurements required high measuringlight intensity which had to be kept off the sample except when actually making the measurement. To get more measuring-light we boosted the voltage on the tungsten projector lamp measuring-light source for a few milliseconds by shorting a resistance in series with it during the measurement. The increased current would have burned the lamp out if continued much longer, and these boostings did shorten the lamp life. Since the lamp temperature rose too slowly at the boosted current level we also arranged to discharge thru the lamp at the beginning of the boost a large capacitor charged to about 100 volts. The voltage was adjusted by trial and error to give a fiat response of the photomultiplier during the measuring period. Incidentally, this system allowed us to compensate for slow changes of sensitivity of the photomultiplier following the turning on of the measuring light. We spent much time studying photomultiplier behavior. Much synchronization circuitry had to be developed to insure that opening the shutter on the measuring-light, boosting the measuring light, triggering the oscilloscope, the laser and the camera all happened at the right times. In the final system one opened the camera, then pressed and held the button to enable the laser, then a button to open the measuring-light shutter, all by hand. Then a photocell sensed the opening of the shutter and triggered the laser, signals from which triggered the scope all with properly adjusted delays. Mr. Armin Weiss helped develop these circuits (DeVault 1964). At the lower temperatures the measurements were slower and we did not boost the lamp. We then used A.C. coupling (series capacitor) of the signal to the scope and waited long enough for the transient produced by opening the shutter to die out before firing the laser. With this system, to get the total signal amplitude (for calculating absorbancy changes) we had to make another measurement with D.C. coupling. Later (1969) we (including Mr. Mark Bilk and Mr. Drew Henderson) developed a much more convenient automatic offset circuit (not published because of referee trouble). Other details of set-up can be found in DeVault (1978). Our measurements were soon repeated by Leslie Dutton, Toru Kihara, James McCray and J.P.

Thornber (1971) on a subchromatophore preparation, on another species, Rhodopsuedomonas sp. N.W., by Kihara and McCray (1973) and again on C vinosum by Brian Hales (1976). Akinori Sarai (Sarai and DeVault 1984) more recently remeasured the whole temperature curve for both high and low potential cytochromes from room temperature to 7K with more accuracy and attention to details. We are still composing a detailed presentation with analysis of biphasicity in the kinetics. The interpretation as "tunneling" harks back to Willard Libby (1940, 1952). He had explained some peculiar chemical phenomena as caused by electron tunneling. So when we were faced with the peculiar temperature dependence of cytochrome photooxidation in C. vinosum it was natural to consider tunneling as a possible explanation. It seemed to fit! We thought in terms of electron tunneling at first. The reasoning went: the slowness of the transfer from cytochrome to Bchl at low temperature indicated a barrier to the transfer. The zero activation energy indicated tunneling of the barrier. Rudolph A Marcus tried to explain to us that the temperature dependence at higher temperature showed involvement of nuclear motion and its absence at lower temperature showed nuclear tunneling. Electron tunneling is still there but the evidence for it is in the preexponential factor of the rate dependence. It took a while for this lesson to sink into my head, I think because I didn't completely understand the mechanism of vibronic coupling to electron transfer until I had a chance to study in detail the papers of Marcus (1965), Hopfield (1974) and Jortner (1976) which I had to do to write my review (DeVault 1980, 1984). In the 1966 paper we had attempted to explain the temperature dependence as the result of thermal vibration of the barrier width for electron tunneling, but the required parameters were unrealistic. So we accepted that the temperature dependent and the temperature independent portions of the curve may represent two different pathways, maybe even two different cytochromes. The latter turned out to be wrong but Bixon and Jortner (1986a, 1986b) are renewing the two pathways idea. The same apparatus was used for a number of early discoveries. Bill Parson showed that P + forms first and is the agent that oxidizes the cytochrome (Parson 1968). Photosynthesis experts, including Lou Duysens and Rod Clayton, had theo-

rized that this is the case but there was still some controversy. Toru Kihara examined many species of bacteria and characterized the low temperature photooxidation of both high and low-potential cytochromes in them (Kihara and Chance 1969). William Hildreth showed that the "520 nm shift" in green plants is carotenoid (Hildreth 1970). Michael Seibert did much to unravel the relationships between the high and low potential cytochtomes and the reaction center in Chromatium (Seibert and DeVault 1970, 1971). Some of the work was paralleled independently by that in Parson's laboratory. Parson also explained Seibert's low potential transient, P424, as carotenoid triplet. We found that high pressure diminished cytochrome photo-oxidation (Chance et al. 1979). Robert Floyd (Floyd et al. 1971) showed that the cytochrome photo-oxidized in green leaves at liquid nitrogen temperature (Chance and Bonner, 1963) was actually cytochrome b-559 and that it was oxidized by electron donation to photooxidized P-680, the Photosystem II reaction center. Toru Kihara and James McCray found that cytochrome oxidation and reduction in many types of systems require the presence of water (Kihara and McCray 1973). When they substituted D20 for H20 the rates were diminished by 40%. Sei Izawa, Ruud Kraayenhof and Enno Ruuge (Izawa et al. 1973) showed that KCN inhibited electron transport in green plant systems at the plastocyanin site between cytochrome f and P700. Wolfgang Junge photodissociated the CO complex of cytochrome c oxidase in various preparations of or from mitochondria with polarized light from the laser. He observed the induced dichroism with polarized measuring light and concluded from a general lack of relaxation of the dichroism in the time observed that the cytochrome oxidase is not mobile in the membrane or the plane of the heme is perpendicular to an axis of mobility (Junge and DeVault 1975). Andrew Rubin and I found that the energy state (ATP level) may determine whether the high-potential or the low potential cytochrome in Chromatium will be oxidized by P + (Rubin and DeVault 1978). This has yet to be confirmed. Mayfair Kung discovered high-order fluorescence (wavelength shorter than that of the

e x c i t i n g light) f r o m p h o t o s y n t h e t i c b a c t e r i a subjected to i n t e n s e l a s e r p u l s e s ( K u n g a n d D e V a u l t 1978). Several rise-times (carotenoid and P+) were m e a s u r e d at s m a l l e r v a l u e s t h a n p r e v i o u s l y k n o w n , to be l a t e r r e d u c e d f u r t h e r by o t h e r s w i t h p i c o s e cond methods. It is a m a z i n g to m e to see t h e v a s t i n c r e a s e in u n d e r s t a n d i n g o f p h o t o s y n t h e s i s o v e r t h e p a s t 50 years. I a m h a p p y to h a v e c o n t r i b u t e d a s m a l l p a r t to it a n d m o s t p l e a s e d t o e n j o y t h e f r i e n d s h i p o f t h e whole photosynthesis community.

References Bixon M and Jortner J (1986a) On the mechanism of cytochrome oxidation in bacterial photosynthesis. Quantum tunnelling effects revisited. FEBS Lett 200:303-308 Bixon M and Jortner J (1986b) Coupling of protein modes to electron transfer in bacterial photosynthesis. J Phys Chem 90: 3795-3800 Chance B and Bonner WD Jr (1963) The temperature insensitive oxidation of cytochromefin green leaves - a primary biochemical event of photosynthesis. In: Photosynthesis Mechanisms in Green Plants, pp 66-81. Nat. Acad. Sci. - Nat Res Council Publ No. 1145 Chance B and DeVault D (1964) On the kinetics and quantum efficiency of the chlorophyll-cytochrome reaction. Berichte der Bunsengesellschaft fiir physikalische Chemie 68:722-726 Chance B and Nishimura M (1960) On the mechanism of chlorophyll-cytochrome interaction: the temperature insensitivity of light induced cytochrome oxidation in Chromatium. Proc Nat Acad Sci USA 46:19-24 Chance B, DeVault D, Legallais V, Mela L and Yonetani T (1967) Kinetics of electron transfer reactions in biological systems. In: Claesson S (ed) Fast Reactions and Primary Processes in Chemical Kinetics (Nobel Symposium 5), pp 437-468. Stockholm: Almqvist & Wiksell Chance B, DeVault D, Tasaki A and Thornber JP (1979) The effects of high hydrostatic pressure on light-induced electron transfer and proton binding in Chromatium. In: Chance B, DeVault D, Frauenfelder H, Marcus RA, Schrieffer JR and Sutin N (eds) Tunneling in Biological Systems, pp. 387-402. New York: Academic Press Chance B, Schleyer H and Legallais V (1963) Activation of electron transfer in a Chlamydomonas mutant by light pulses from an optical maser. In: Japanese Soc. Plant Physiol (eds). Studies on Microalgae and Photosynthetic Bacteria, Special Issue of Plant and Cell Physiology, pp. 337-346. Univ. of Tokyo Press DeVault D (1978) Nanosecond absorbance spectrophotometry. Methods in Enzymology 54:32-46 DeVault D, (1964) Photochemical activation apparatus with optical maser. In: Rapid Mixing and Sampling Techniques in Biochemistry, pp 165-174. New York: Academic Press

DeVault D (1980) Quantum mechanical tunnelling in biological systems. Quart Rev Biophys 13:387-564 DeVault D (1984) Quantum Mechanical Tunnelling in Biological Systems. Cambridge: Cambridge University Press DeVault D (1987) Science and Satyagraha. Published privately by D.DeVault, 1206 Northwood Dr.N., Champaign, I161821. 102 + vii pages, $7.50 + $1.00 postage and handling DeVault D and Chance B (1966) Studies of photosynthesis using a pulsed laser: I. Temperature dependence of cytochrome oxidation rate in chromatium. Evidence for tunneling. Biophys. J 6:825-847 DeVault D, Parkes JH and Chance B (1967) Electron tunnelling in cytochromes. Nature 215:642-644 Dutton PL, Kihara T, McCray JA and Thornber JP (1971) Cytochrome C-553 and bacteriochlorophyll interaction at 77 K in chromatophores and a subehromatophore preparation from Chromatium D. Biochim Biophys Acta 226:81-87 Floyd RA, Chance B and DeVault D (1971) Low temperature photoinduced reactions in green leaves and chloroplasts. Biochim Biophys Acta 226:103-112 Hales BJ (1976) Temperature dependency of the rate of electron transport as a monitor of protein motion. Biophys J 16: 471-480 Hildreth W (1970) The 520 nm absorption change in barley and a chlorophyll b-deficient mutant. Arch Biocbem Biophys 139: 1-8 Hopfield JJ (1974) Electron transfer between biological molecules by thermally activated tunneling. Proc Nat Acad Sci USA 71:3640-3644 Izawa S, Kraayenhof R, Ruuge EK and DeVault D (1973) The site of KCN inhibition in the photosynthetic electron transport pathway. Biochim Biophys Acta 314:328-339 Jortner J (1976) Temperature dependent activation energy for electron transfer between biological molecules. J Chem Phys 64:4860-4867 Junge W and DeVault D (1975) Symmetry, orientation and rotational mobility in the a 3 heine of cytochrome c oxidase in the inner membrane of mitochondria. Biochim Biophys Acta 408:200-214 Kamen MD (1974) The birthplace of big science. Bull At Sci 30: 42-46 Kamen MD (1986) A cupful of luck, a pinch of sagacity. Ann Rev Biocbem 55:1-34 Kihara T and Chance B (1969) Cytochrome photo-oxidation at liquid nitrogen temperatures in photosynthetic bacteria. Biochim Biophys Acta 189:116-124 Kihara T and McCray JA (1973) Water and the cytochrome oxidation-reduction reactions. Biochim Biophys Acta 292: 297-309 Kung MC and DeVault D 0978) High-order fluorescence and excitation interaction in photosynthetic bacteria. Biochim Biophys Acta 501:217-231 Libby WF (1940) Reactions of high energy atoms produced by slow neutron capture. J Am Chem Soc 62:1930-1943 Libby WF (1952) Theory of electron exchange reactions in aqueous solution. J Phys Chem 56:63-68 Marcus RA (1965) On the theory of electron-transfer reactions. VI. Unified treatment for homogeneous and electrode reactions. J Chem Phys 43:679-701

10 Parson W (1968) The role of P870 in bacterial photosynthesis. Biochim Biophys Acta 153:248-259 Ruben S, Kamen MD, Hassid WZ and DeVault DC (1939) Photosynthesis with Radio-Carbon. Science 90:570-571 Rubin AB and DeVault D (1978) The effects of uncoupler on the rate of cytochrome oxidation and reduction in the photosynthetic bacterium, Chromatium. Evidence for a possible cytochrome switching. Biochim Biophys Acta 501:440--448 Sarai A and DeVault D (1984) Temperature dependence of

high-potential cytochrome photoxidation in C. vinosum. In: Sybesma C (ed) Advances in Photosynthesis Research, Vol. I, pp 653-656. The Hague: Nijhoff/Junk Seibert M and DeVault D (1970) Relations between the laserinduced oxidations of the high and low potential cytochromes of Chromatium D. Biochim Biophys Acta 205:220-231 Seibert M and DeVault D (1971) Photosynthetic reaction center transients, P435 and P424 in Chromatium D. Biochim Biophys Acta 253:396-411