Electron Movement Through Proteins and DNA

Free Radical Biology & Medicine, Vol. 22, No. 7, pp. 1271–1276, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/97 $17.00 / .00

PII S0891-5849(96)00548-5

Review Article ELECTRON MOVEMENT THROUGH PROTEINS AND DNA Martyn C. R. Symons School of Applied Sciences, Department of Chemistry, De Montfort University, The Gateway Leicester, Leicester LE1 9BH, UK (Received 9 February 1996; Accepted 16 October 1996)

Abstract—Nature utilizes the phenomenon of single electron transfer very widely, especially in metallo-proteins. In systems when the metal donor (D) is well separated from the acceptor (A) by polypeptide chains, the transferring electron is presumed to be bonded, in part, by these chains, which may influence the pathway taken. This situation can be probed by radiolytic injection of electrons into proteins at low temperatures. One aim of this brief review is to consider how information derived from such radiolysis studies, and followed by ESR spectroscopy, may possibly impinge on studies of D-A systems. Electrons can also be injected into duplex DNA in this way, and the results are compared with those for proteins. They are also considered in the light of recent studies of D-A electron-transfer via a polynucleotide strand. It seems that such transfers are very efficient, and it is tentatively suggested that Nature may also use this conductivity in some as yet undiscovered systems. q 1997 Elsevier Science Inc. Keywords—Electrons, Proteins, DNA, Electron spin resonance, Free radicals

the protein structure is surely involved. The precise mechanism remains a matter that is debated. The chemically important point is that electron-transfer is much faster than the potential proton transfers that would efficiently trap the electron at an amido site. It is possible that electron movement through DNA is also of importance, although I am not aware of any examples as yet. Perhaps I can postulate that electron migration does occur ‘‘intentionally’’ in circumstances yet unknown? Ionizing radiation certainly induces electron migration over considerable distances within the stacked bases.

INTRODUCTION

Efficient movement of electrons from an electron-donor (D) to an electron-acceptor (A) site is a key aspect of the reactions of many enzymes. Hence, the ways in which electrons move through protein strands has attracted wide attention. In many cases, these centers are close enough for the electrons to transfer directly from one center to the other in a tunnelling process, in which case movement is extremely rapid. My concern herein is with the relatively slow processes that occur when the D and A centers are far apart, and movement via

AIMS

My B.Sc and Ph.D. degrees are from London University via Battersea Polytechnic, where I studied and taught for several years, with a 3-year gap for national service in the army. I moved to Southampton University in 1953 as a lecturer in organic chemistry, then to Leicester University in 1960 as Professor of Physical Chemistry. After a brief spell at Essex University as a Research Professor in Chemistry, I am now a visiting Professor in the School of Applied Sciences at De Montfort University Leicester and in the Bone and Joint Unit of the London Hospital Medical College. Current work centers on (i) the study of radiation processes in chemistry and biology using, primarily, EPR Spectroscopy as a probe, and (ii) Spectroscopic studies of solvation mainly with water as one component. These and related studies are described in over 1000 papers that I have written over the years. My nonscientific interests are landscape water-color painting, and piano playing (especially Chopin).

My main aim is to endeavor to establish links between ‘‘conventional’’ studies of electron migration in these biopolymers and studies based on the use of ionizing radiation. Although the work based on D-A systems in proteins, both natural and primed with added donor units, has been very widely reviewed, they have not, so far as I am aware, been considered in the light of radiation studies. It, therefore, seemed to be worthwhile to give a brief outline of these D-A systems and to try to show 1271

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how the results of radiation studies may be complimentary to these key systems. Also in the DNA area, we and others have presented results for extensive electron migration along the stacked bases. It seemed of interest to compare those results with a recent study of electron transfer using a D-A system. ELECTRON MIGRATION IN PROTEINS

Nature uses electron transfer reactions extensively, presumably because of their simplicity and great speed. These generally occur within complex enzyme systems, such as, for example, the trans-membrane mitochondrial cytochrome oxidase enzymes. For this reason, a range of systems have been studied kinetically with a view to probing the detailed mechanisms involved. These studies have been reviewed widely.1–3 All I wish to do herein is to summarize the types of experiments that are used, and not to present a further review on the topic, nor to comment on the details of current theories. In many studies, metallo proteins are modified by the addition of a transition metal complex to a histidine or other basic group on the periphery of the protein. Ru(III) complexes are ideal for this purpose. Flash photolysis or chemical methods are used to form the Ru(II) derivative rapidly, and then e-transfer from Ru(II) to the indigenous metal center, and possibly the reverse, can be followed kinetically. The results have led some to conclude that specific pathways for e-transfer are present in proteins containing (D) and (A) sites that are well separated from each other. This implies that some regions of proteins are better conductors than others, and that these regions comprise ‘‘linear’’ pathways between D and A units. Results for a variety of Ru-labeled systems, reported by Gray and his co-workers1,4,5 have been nicely accommodated by a theoretical model for tunnelling in which covalent bonds, hydrogen bonds, and free space are included explicitly.6 However, it seems that this evidence is not compelling.7 One problem is that of defining the distance through which the electron has to move. One limit is the metal-metal separation, and another is the edge separation between the nearest ligands. It seems that the ‘‘preferred pathway’’ concept is not required if these distances are estimated by a different procedure. Hence, this interesting controversy remains unsolved.8 One of the most favored theoretical treatments is known as the ‘‘superexchange’’ formalism.9–11 Others include path integral methods,12 and pathway analysis.13 All methods are, perforce, no more than very approximate, because of the huge number of units involved. It is remarkable that all these methods

appear to be reasonably successful, suggesting that very detailed descriptions may not be necessary. Even so, it is important to realise that there is always some degree of adjustment to the final computed data. From the point of view of this review, the important result is that theory and experiment agree that there are, indeed, conducting pathways through which electrons, en route from donor to acceptor, can move with very high speeds. The key, so far as nature is concerned, is speed. This is also the conclusion arrived at from radiation studies, which serve to illustrate the limiting situation in which the ‘‘donor’’ is, in effect, left out. PROTEINS AND IONIZING RADIATION

We have developed a low-temperature radiolytic method, which, in my view, also explores the movement of electrons through proteins. This work was initiated by our discovery that low-temperature irradiation of oxyhemoglobin, HbFeO2, gave a remarkably high yield of the novel center, HbFeO2 0,14,15 which was unambiguously characterized by electron spin resonance spectroscopy (ESR—this term and electron paramagnetic resonance spectroscopy—EPR—are synonymous). This was interpreted in terms of rapid migration of the ejected electrons along the protein strands, with specific trapping at the haem (FeO2) units. Using the ferric derivative, a similar conversion, to the deoxyhemoglobin, was observed.16,17 In contrast, the electron loss centers were trapped directly, without significant migration, and hence, these do not interfere with electron transfer studies. The mechanism for direct radiation damage to proteins that we have proposed, is summarized in Fig. 1. At 77 K the major electron loss centers generated by ionizing radiation were shown to be amido radicals, formed by N-H proton loss from the radical cations. We know that the holes do not migrate extensively prior to being trapped, because electron-rich centers present in certain proteins do not lose electrons. For example, FeII is not converted into FeIII in detectable yields in deoxy Hb.17 Hence, the representation in Fig. 1b is reasonable. The ejected electrons might have reacted similarly, being trapped at any of the backbone amide units. However, they only react at these ubiquitous sites in the absence of sites with higher electron affinities. Thus, they migrate rapidly through the protein and are captured efficiently at acceptor sites. This means that a delocalized ‘‘conduction band’’ description is appropriate, which is just the conclusion drawn in E.T. studies. When more than one electron-affinic site is present,

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which must be indiscriminate, yields thermalized electrons that migrate very rapidly over long distances, throughout the protein, and are selectively captured at acceptor sites, such as Fe(III), Cu(II),19 or RS-SR units.20 In contrast, the majority of the hole centers seem to migrate to nearby amido groups and become trapped by proton loss without undergoing extensive migration.21 Thus, these centers will occupy different sites in different molecules, and can be any distance from the A-sites. These are considered to be of no major significance to the problem of electron transfer. What do these results reveal? It seems to me that there are two factors that need to be considered, i) pathways, and ii) systems.

Pathways

Fig. 1. Model for electron transfer in a section of protein: In (a), the electron gain center is depicted. In this case, electron transfer between amido units is faster than proton transfer. Hence, excess electrons can migrate rapidly throughout the protein, and this is no longer a meaningful representation. In (b), transfer of the electron loss center is depicted. (This is ‘‘hole’’ transfer; electron transfer is in the reverse direction.) This is rapidly prevented by proton transfer to give an amido-radical, detected by ESR spectroscopy. (The dots, ...., represent hydrogen bonds.)

an ejected electron may add statistically, or selectively, depending upon the magnitude of any barrier that may exist. If two A centers (A1 and A2) are close enough, it may add statistically but the electrons on A1 may then move to A2 even at 77 K. For example, for xanthine oxidase, capture initially occurred at one of the iron-sulphur centers (FeSI). However, after long exposures, capture at the MoVI units was clearly detected. Probably capture occurs at both, but further transfer from MoV to (FeSI) is rapid even at 77 K. On annealing, transfer occurred from the (FeSI) cluster to the second cluster (FeSII). This is the unit that is preferentially reduced at room temperature, so it is thermodynamically the most stable, but kinetically the least reactive of these three centers.18 However, for hemoglobin containing both FeIII iron and FeO2 units, capture seems to be indiscriminate at 77 K, giving FeII and FeO2 0 centers statistically. No transfer from the ‘‘unstable’’ FeO2 0 units to FeIII centers occurred at 77 K, even after long storage.14,15 Thus our results suggest that initial electron ejection,

When discussing our results,21 we used a pictorial model in which the electron moved very rapidly along the protein backbone, the contact time with any one local unit being far too short for any trapping to occur. Each amido p-system is separated from its neighbors by a single saturated carbon. It is well established that delocalization readily occurs via such units,22 and, hence, the system may well embrace many amido groups that are favorably located. This model assumes that the p*-s*M.O. of the ubiquitous amido units is favored over any form of pure s*-delocalization. It is clearly oversimplified, because migration may well occur preferentially across the hydrogen-bonded amido links, thereby bypassing the —CHR— units.23 Presumably both pathways need to be considered. Clearly, there should be a significant differentiation between helical, b-pleat and random regions of a protein.23 Furthermore, trapping at amido or other sites must sometimes occur if, for example, a proton is already partially transferred as a result of vibrational movement, or the favored pyramidal structure is fortuitously formed as the electron ‘‘flirts’’ with a specific amido group. These factors may well also come into play for long-distance transfer in redox enzymes and pathways that might trap the electron at undesirable sites are probably avoided in nature. I imagine that our results may be important in that they represent a limit in which there is no meaningful direct overlap between D and A units. As the separation between A and D increases, so the participation of this upper limit must increase. Ultimately, the barrier becomes the ability of D to inject an electron into the protein. It will then find its target, but may be diverted to others, if they are present. In this limit then is probably no ‘‘pathway’’ to any specific acceptor, and the electron is free to explore the whole protein.

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Systems I suggest that this type of study is complementary to the systems involving added metal ion units. Our systems have two advantages, namely that by working at low temperatures it is possible to form and trap electron-rich centers and then to study their reactions on annealing. Thus natural unmodified systems can be studied directly. It is not necessary to add transition metal ions or other electron donors or acceptors to the system, thereby avoiding the possibility of introducing perturbations. My hope is that this relatively simple method will be exploited in future studies of electron transfer processes. I now turn to similar studies using DNA.

DNA AND IONIZING RADIATION

The effects of ionizing radiation on DNA in the presence of water at low temperatures are much simpler than might be expected.24–26 These systems have been studied by ESR spectroscopy and the results show unequivocally that the final, trapped, electron loss and electron gain centers are localized in the bases, the former being mainly Gi/ centers and the latter Ti0 or Ci0 centers, trapped by proton loss or proton gain. Although they are generated together, the hole and electron centers that are detected must have moved apart. Some recombine with light emission,27 but none remain trapped on adjacent bases, because the g Å 4 feature, characteristic of pair-trapped radicals,28 has never been seen in the ESR spectra. For DNA, electron ejection must be from any site, including the sugar and phosphate units, and solvating water.29 However, ESR results suggest that both centers migrate rapidly into the base p-systems without being trapped. Once in the stacks, both centers can move. In one limit one can postulate valence and conduction bands, inducing ‘‘linear’’ delocalization directly. However, both centers are readily trapped (as established, e.g., by conductivity studies)29 probably by proton transfer.24 Normally, electron transfer is so fast that such relaxation processes leading to trapping only rarely compete. In the absence of any added traps, the quantum yields (G-values) of trapped centers are in the normal range. The hole centers are trapped very rapidly by proton loss. However, the electrons move extensively (see below), before being permanently trapped to give Ti0 or Ci0 centers, in our view as a result of protonation. On incorporation of an electron-affinic intercalator into the base stacks there was a dramatic reduction in the yields of DNA radical anions (characterized by the doublet shown in Fig. 2) and a concomitant growth of

a singlet feature identical in g-value and width with the ESR spectrum for the intercalator radical anions at 77 K (Fig. 2b).30 (These were prepared unambiguously in a methanol glass for comparison.) Further support for this scheme came from annealing studies. On warming DNA irradiated at 77 K, the Ti0 centers are converted largely into iTH centers (see insert I), which have a characteristic octet ESR signal that can be fully quantified. Yields of iTH were also dramatically reduced on the incorporation of an intercalator, falling, for example, to about 40% of the normal value for an intercalator concentration ratio of 1:150 base pairs. Thus, it seems certain that ejected electrons are efficiently trapped, but only at the pyrimidine bases. The key question is, how far and how fast do electrons move, prior to capture? In our view, the electrons travel extremely rapidly and can move long distances along the base stacks prior to being captured. Because this is by no means universally accepted, I present a brief description of the results. These are summarized in Fig. 3, which shows the

Fig. 2. First derivative ESR spectra for aqueous DNA after exposure to ionizing radiation at 77 K. (a) In the absence of intercalator, showing the characteristic doublet assigned to a mixture of Ci0 and Ti0 (their spectra are almost identical. (b) After the addition of an intercalator (1:75 base pairs) showing the loss of the doublet and gain of an intense narrow singlet, assigned to the intercalator anion. the asterisks indicate shoulders from the cation centers.

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Similarly, Anderson et al.32 are not studying the same type of migration as we are. They use pulse-radiolysis at room temperature, so it is addition of aquated electrons (eaq 0) that they study. Aquated electrons react quite slowly and selectively with duplex DNA. Probably they only add to ‘‘favorable’’ bases that are able to trap them directly. Certainly, they clearly cannot migrate far once they have entered the base stack and they probably rarely, if ever, enter the ‘‘conductionband.’’ Hence, intercalated molecules are not efficient at capturing these solvated electrons via migration over long distances. D-A ELECTRON TRANSFER IN DNA Fig. 3. Dependence of the concentration of intercalator radical anions relative to that of DNA radical anions (y), on the relative concentration of the intercalator (l). The full line is for the equation y Å l0tanh 2bl, where b is the mean distance traversed by the electron.

decrease in concentration of the DNA electron centers as the concentration of electron-affinic intercalators are increased.30 [For y, the limit of 1.00 is pure DNA anionradicals and the limit of 0.00 is for pure intercalator anions. The relative concentrations (l) are expressed as the fraction of intercalator molecules per base pair of the duplex DNA.] The results were analyzed statistically, and were shown to fit well with the predicted pattern, assuming random distribution of the intercalator. This analysis showed that there was a 50% probability of forming the intercalator anion rather than the DNA anions for a concentration ratio of 1:110 base pairs. Electron capture by the intercalator was almost complete at ca. 1:30 base pairs. The two intercalators used are shown in insert II. It is noteworthy that at pK7 these are dications. Even in the absence of an intercalating function these are strongly attracted to DNA, which has the effect of greatly enhancing the extent of intercalation. In our low temperature studies we assumed that intercalation was effectively complete.30 It has been inferred that our results conflict with those of Fielden, O’Neill, and co-workers,31 and of Anderson and co-workers.32 In our view, we are all measuring different things and the results are not really contradictory. Thus, the former group study mainly the emission of light from the singlet excited state of G following electron ejection and very rapid return.31 Thus, in this case, the significant distance is how far the electron moves, on average, before returning. We look only at those that do not return, and estimate that this is the situation for a large majority of the electrons. So, there is no comparison.

Rapid migration of an electron through a short DNA oligomer has recently been demonstrated by Barton and her co-workers.33 In a beautifully designed experiment, two metal complexes were covalently linked to the ends of the oligomer in such a way that one of the ligands clearly intercalated into the base stacks at either end. Photo-induced electron transfer is envisaged to occur via the intercalated p-ligands through the p-system of the stacked bases. In these studies only electron transfer was observed. For the relatively short strands used (15 base pairs), our results suggest that capture by C or T would be improbable. However, for longer strands it would be most interesting to see if such photolyses result in the formation of some Ci0 and Ti0 centers. This could be tested at 77 K using ESR spectroscopy, or by strand break studies after room temperature photolysis. More recently, a comparable study, using tethered ruthenium complexes, has led to similar conclusions.34 Finally, to join the two studies together, it is interesting to note that there appears to be some electron leak from histone proteins to DNA on irradiation of chromatin and cell nuclei.35 This implies that there is a real ability for electrons to move out of the protein strands, bypassing the sugar-phosphate units and moving into the base p-system. CONCLUSIONS

It is clear that electrons can migrate over long distances in both proteins and DNA, without being trapped. In the absence of specific acceptors, ultimate trapping probably involves proton transfer. For DNA there is a clear pathway, which does not depend on specific sequences of bases. For proteins it is still not clear if nature provides specific pathways in enzymes whose function is to move electrons rapidly from one site to another. It is suggested that radiolysis at low

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temperature is a very good method for ‘‘setting the electron transfer clock’’ in such proteins, thus making it easier to study natural systems. Acknowledgements — I thank all my past collaborators for their invaluable help, and the Cancer Research Campaign, the Ministry of Defence, the Leverhume Trust and the Association for International Cancer Research for financial assistance, over the years.

19. 20. 21. 22.

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ABBREVIATIONS

ESR—electron spin resonance D-A—electron donor-electron acceptor units (Fe-S)—iron-sulphur clusters in proteins RS-SR—dialkyl disulphides T, C, G, A—DNA bases, thyamine, cytosine, guanine, and adenine

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