A model for photoreceptor-based magnetoreception in birds

A large variety of animals has the ability to sense the geomagnetic field and utilize it as a source of directional (compass) information. It is not known by which biophysical mechanism this magnetoreception is achieved. We investigate the possibility that magnetoreception involves radical-pair processes that are governed by anisotropic hyperfine coupling between (unpaired) electron and nuclear spins. We will show theoretically that fields of geomagnetic field strength and weaker can produce significantly different reaction yields for different alignments of the radical pairs with the magnetic field. As a model for a magnetic sensory organ we propose a system of radical pairs being 1) orientationally ordered in a molecular substrate and 2) exhibiting changes in the reaction yields that affect the visual transduction pathway. We evaluate three-dimensional visual modulation patterns that can arise from the influence of the geomagnetic field on radical-pair systems. The variations of these patterns with orientation and field strength can furnish the magnetic compass ability of birds with the same characteristics as observed in behavioral experiments. We propose that the recently discovered photoreceptor cryptochrome is part of the magnetoreception system and suggest further studies to prove or disprove this hypothesis.     

Role of exchange and dipolar interactions in the radical pair model of the avian magnetic compass

It is not yet understood how migratory birds sense the Earth's magnetic field as a source of compass information. One suggestion is that the magnetoreceptor involves a photochemical reaction whose product yields are sensitive to external magnetic fields. Specifically, a flavin-tryptophan radical pair is supposedly formed by photoinduced sequential electron transfer along a chain of three tryptophan residues in a cryptochrome flavoprotein immobilized in the retina. The electron Zeeman interaction with the Earth's magnetic field (∼50 μT), modulated by anisotropic magnetic interactions within the radicals, causes the product yields to depend on the orientation of the receptor. According to well-established theory, the radicals would need to be separated by >3.5 nm in order that interradical spin-spin interactions are weak enough to permit a ∼50 μT field to have a significant effect. Using quantum mechanical simulations, it is shown here that substantial changes in product yields can nevertheless be expected at the much smaller separation of 2.0 ± 0.2 nm where the effects of exchange and dipolar interactions partially cancel. The terminal flavin-tryptophan radical pair in cryptochrome has a separation of ∼1.9 nm and is thus ideally placed to act as a magnetoreceptor for the compass mechanism.     

The horizontal magnetic dance of the honeybee is compatible with a single-domain ferromagnetic magnetoreceptor

Although honeybees are able to sense the geomagnetic field, very little is known about the method in which they are able to detect it. The recent discovery of biochemically precipitated magnetite (Fe₃O₄) in bees, however, suggests the possibility that they might use a simple compass organelle for magnetoreception. If so, their orientation accuracy ought to be related to the accuracy of the compass, e.g. it should be poor in weak background fields and enhanced in strong fields. When dancing to the magnetic directions on a horizontal honeycomb, bees clearly show this type of alignment behavior. A least-squares fit between the expected alignment of a compass and this horizontal dance data is consistent with this hypothesis, and implies that the receptors have magnetic moments of 5 × 10^?13 emu, or magnetite volumes near 10^?15 cm³. Additional considerations suggests that these crystals are slightly sub-spherical and single-domain in size, held symmetrically in their receptors, and have a magnetic orientation energy of approximately to 6 kT in the geomagneticfield. A model of a magnetite-based magnetoreceptor consistent with these constraints is discussed.     

Magnetoreception

The vector of the geomagnetic field provides animals with directional information, while intensity and/or inclination provide them with positional information. For magnetoreception, two hypotheses are currently discussed: one proposing magnetite-based mechanisms, the other suggesting radical pair processes involving photopigments. Behavioral studies indicate that birds use both mechanisms: they responded to a short, strong magnetic pulse designed to change the magnetization of magnetite particles, while, at the same time, their orientation was found to be light-dependent and could be disrupted by high-frequency magnetic fields in the MHz range, which is diagnostic for radical pair processes. Details of these findings, together with electrophysiological and histological studies, suggest that, in birds, a radical pair mechanism located in the right eye provides directional information for a compass, while a magnetite-based mechanism located in the upper beak records magnetic intensity, thus providing positional information. The mechanisms of magnetoreception in other animals have not yet been analyzed in detail.     

Magnetic Compass of Birds Is Based on a Molecule with Optimal Directional Sensitivity

The avian magnetic compass has been well characterized in behavioral tests: it is an “inclination compass” based on the inclination of the field lines rather than on the polarity, and its operation requires short-wavelength light. The “radical pair” model suggests that these properties reflect the use of specialized photopigments in the primary process of magnetoreception; it has recently been supported by experimental evidence indicating a role of magnetically sensitive radical-pair processes in the avian magnetic compass. In a multidisciplinary approach subjecting migratory birds to oscillating fields and using their orientation responses as a criterion for unhindered magnetoreception, we identify key features of the underlying receptor molecules. Our observation of resonance effects at specific frequencies, combined with new theoretical considerations and calculations, indicate that birds use a radical pair with special properties that is optimally designed as a receptor in a biological compass. This radical pair design might be realized by cryptochrome photoreceptors if paired with molecular oxygen as a reaction partner.     

Exploring the possibilities for radical pair effects in cryptochrome

The ability of some animals to sense magnetic fields has long captured the human imagination. In our recent paper, we explored how radical pair effects in the protein cryptochrome may underlie the magnetic orientation sense of migratory birds. Here we explain our model and discuss its relationship to experimental results on plant cryptochromes, as well as discuss the next steps in refining our model, and explore alternate but related possibilities for modeling and understanding cryptochrome as a magnetic sensor.Key words: cryptochrome, radical pair machanism, avian orientation, magnetic field effect, Arabidopsis thaliana, avian magnetoreception, magnetic sensorThe ability of some animals to sense magnetic fields is a long-standing open problem in biology. Over the past 50 years, scientific studies have shown that a wide variety of living organisms have the ability to perceive magnetic fields and can use information from the earth''s magnetic field in orientation behavior. The best-studied example of animal magnetoreception is the case of migratory birds, who use the earth''s magnetic field, as well as a variety of other environmental cues, to find their way during migration.The two prevailing hypotheses for the mechanism of avian magnetoreception are an iron-mineral-based explanation, wherein birds use small deposits of magnetic iron minerals^1,2,12 in the base of their beaks for magnetic orientation, and a radical-pair-based explanation, in which a magnetically sensitive chemical reaction in the eye of the bird enables perception of the magnetic field via its effects on reaction products. The latter hypothesis is based on the idea that a radical pair reaction may take place in the protein cryptochrome in the retina of the bird.^3,4 Cryptochrome contains a blue-light-absorbing chromophore, flavin adenine dinucleotide (FAD); this FAD cofactor is reduced via a series of light-induced electron transfers from a chain of three tryptophans that bridge the space between FAD and the protein surface (see Fig. 1). The hypothesis explored in our paper⁴ is that a radical pair reaction takes place between FAD and the tryptophans in the photoreduction pathway which modulates the signaling activity of cryptochrome. The specifics of this idea are outlined in Figure 1.Open in a separate windowFigure 1Right: Cryptochrome internally binds the FAD cofactor and contains a three-tryptophan photoreduction pathway conserved from photolyase, consisting of Trp400, Trp377, and Trp324, with Trp400 nearest the FAD and Trp324 closest to the protein surface. After the FAD cofactor absorbs a photon, bringing it into an excited state, it is protonated from a nearby acidic residue, and then electron transfer proceeds from Trp400. At this stage, the semireduced FADH and Trp400⁺ comprise a radical pair—that is, each partner has an unpaired electron, and the spins of those electrons are in a correlated state. Cryptochrome is thought to be in its active, signaling state when the FAD cofactor is in this semireduced FADH form. An electron is then transferred from Trp377 to Trp400 and from Trp324 to Trp377, forming radical pairs FADH + Trp377⁺ and FADH + Trp324⁺ in the process. The Trp324 radical is then deprotonated. Before this final deprotonation, it is possible for the electron to back transfer from the tryptophan to FADH. If this occurs, FADH reverts to the oxidized FAD form, and cryptochrome is no longer in its active state. Left: This schematic of the electron transfer pathway in cryptochrome shows the estimated lifetimes of each of the radical pair states. The system spends most of its time in the FADH + Trp324 radical pair state. Also shown are the electron and nuclear spins on the FADH and Trp324 radicals. Each nuclear spin adds a small contribution to the local magnetic field. The unpaired electron spins are shown here in the singlet (antiparallel) state. They precess around the local magnetic field, which consists of contributions from the external field and from each of the nuclear spins, causing interconversion to the triplet (parallel) state and back again. This singlet-triplet interconversion is the basis of the radical pair effect in the following sense. Electron back-transfer from Trp324 to FADH proceeds only when the unpaired electrons on each radical are in the singlet state. Cryptochrome remains in its active state so long as this back-transfer is impeded. Therefore, singlet-triplet interconversion influences the time cryptochrome can spend in its active state, and so this magnetic-field-driven effect can alter the protein''s signaling behavior.That magnetic field effects do occur in cryptochrome is supported indirectly by experiments done by Margaret Ahmad and co-workers, as reported in their recent paper⁵ on the effects of magnetic fields on cryptochrome-dependent responses in Arabidopsis thaliana seedlings. In our paper, Magnetic Field Effects in Arabidopsis thaliana Cryptochrome-1 (4), we sought to evaluate this possibility computationally, to see whether a magnetic field effect in the FADH - tryptophan radical pair is reasonable. We found that it is possible to see a change in cryptochrome activation yield (the amount of time cryptochrome stays in its active state) of about 10%.Unfortunately, the magnetic field dependence of cryptochrome activation seen in our calculations cannot be taken as exact because of several limitations. Chief among these are that the models of the radical pair did not include all nuclei, and the hyperfine coupling constants were taken from DNA photolyase, which is a protein highly similar to cryptochrome in structure, but which does not necessarily have precisely the same hyperfine coupling for the FAD cofactor and the tryptophans in the photoreduction pathway as does cryptochrome. However, the suggested theory is general and with the knowledge of correct hyperfine coupling constants for the radical pair partners it can be used to calculate the activation yield precisely. Although it would be ideal to obtain hyperfine parameters from experiment, it is also possible to calculate the hyperfine coupling constants with advanced ab initio techniques using the Gaussian package.⁶ Our preliminary calculations of the hyperfine couplings in tryptophan radicals compare well with the values used in our paper.⁴ This sort of calculation creates the opportunity not only to refine our current picture of the radical pair mechanism in cryptochrome, but also to explore other possible radical pairs in the system.In light of work being done by Margaret Ahmad and co-workers (not yet published), it has been suggested recently that the radical pair reaction in cryptochrome may not occur between the FAD cofactor and tryptophan, but in some other radical pair within the protein. It is possible that rather than occurring in the FAD photoreduction process, the radical pair reaction actually takes place in the reoxidation reaction wherein the semireduced FADH is brought back to the oxidized FAD form. One possible radical pair in the back reaction is between FAD and an oxygen molecule which is thought to be involved in the reoxidization process. This radical pair is of particular interest because an oxygen radical would be devoid of hyperfine interactions. Such a radical pair, where one radical has no hyperfine coupling, would be consistent with studies on the effects of weak radio-frequency oscillating magnetic fields on migratory bird orientation. Thorsten Ritz and co-workers found that appropriate orientation behavior depended not only on the strength and angle of the oscillating field, but also that the minimum field strength necessary to disrupt orientation depended on the frequency of the oscillating field in a resonance-like behavior that would be predicted by just such a radical pair^7–9 (personal communication with T. Ritz).The scientific community is still a long way from a complete understanding of avian magnetoreception. The best that may be said of our understanding of it is that birds do demonstrably perceive and use magnetic field information, and that their responses to magnetic fields under different conditions—light intensity and color, magnetic field strength and presence and frequency of oscillating fields—belies a complex phenomenon which is probably the result of multiple receptors which interact in unknown ways.^10,11 However, disorientation responses to low-intensity oscillating magnetic fields are strongly suggestive of the involvement of a radical-pair mechanism, making the exploration of radical pair effects in cryptochrome a useful endeavor. Much remains to be done. Even if cryptochrome is confirmed as magnetoreceptor, it remains for biologists to determine how its signaling modulation enters into a bird''s sensory perception and ultimately its orientation behavior. Nevertheless, radical pair effects in cryptochrome seem promising as a possible source of magnetoreception in birds, and continued investigation may yet shed light on this complex behavior.     

Interaction of magnetite-based receptors in the beak with the visual system underlying 'fixed direction' responses in birds

Background

European robins, Erithacus rubecula, show two types of directional responses to the magnetic field: (1) compass orientation that is based on radical pair processes and lateralized in favor of the right eye and (2) so-called 'fixed direction' responses that originate in the magnetite-based receptors in the upper beak. Both responses are light-dependent. Lateralization of the 'fixed direction' responses would suggest an interaction between the two magnetoreception systems.

Results

Robins were tested with either the right or the left eye covered or with both eyes uncovered for their orientation under different light conditions. With 502 nm turquoise light, the birds showed normal compass orientation, whereas they displayed an easterly 'fixed direction' response under a combination of 502 nm turquoise with 590 nm yellow light. Monocularly right-eyed birds with their left eye covered were oriented just as they were binocularly as controls: under turquoise in their northerly migratory direction, under turquoise-and-yellow towards east. The response of monocularly left-eyed birds differed: under turquoise light, they were disoriented, reflecting a lateralization of the magnetic compass system in favor of the right eye, whereas they continued to head eastward under turquoise-and-yellow light.

Conclusion

'Fixed direction' responses are not lateralized. Hence the interactions between the magnetite-receptors in the beak and the visual system do not seem to involve the magnetoreception system based on radical pair processes, but rather other, non-lateralized components of the visual system.     

Two different types of light-dependent responses to magnetic fields in birds

A model of magnetoreception proposes that the avian magnetic compass is based on a radical pair mechanism, with photon absorption leading to the formation of radical pairs. Analyzing the predicted light dependency by testing migratory birds under monochromatic lights, we found that the responses of birds change with increasing intensity. The analysis of the orientation of European robins under 502 nm turquoise light revealed two types of responses depending on light intensity: under a quantal flux of 8.10(15) quanta m(-2) s(-1), the birds showed normal migratory orientation in spring as well as in autumn, relying on their inclination compass. Under brighter light of 54.10(15) quanta m(-2) s(-1), however, they showed a "fixed" tendency toward north that did not undergo the seasonal change and proved to be based on magnetic polarity, not involving the inclination compass. When birds were exposed to a weak oscillating field, which specifically interferes with radical pair processes, the inclination compass response was disrupted, whereas the response to magnetic polarity remained unaffected. These findings indicate that the normal inclination compass used for migratory orientation is based on a radical-pair mechanism, whereas the fixed direction represents a novel type of light-dependent orientation based on a mechanism of a different nature.     

Why animals respond to the full moon: Magnetic hypothesis

The geomagnetic field is typically about 50 μT (range 20-90 μT). Geomagnetic activity generally decreases by about 4% for the seven days leading up to a full moon, and increases by about 4% after the full moon, lasting for seven days. Animals can clearly detect the changes in magnetic field intensity that occur at full moon, as it has been shown that variations of just a few tens of nT are adequate to form a useful magnetic ‘map’. We think that moonlight increases the sensitivity of animals' magnetoreception because the radical pair model predicts that magnetoreception is light dependent. In fact, there have been some reports of changes in the sensitivity of magnetoreception with lunar phase. We propose a hypothesis that animals respond to the full moon because of changes in geomagnetic fields, and that the sensitivity of animals' magnetoreception increases at this time.     

鸟类磁感受的生物物理机制研究进展

行为学实验表明,许多鸟类能够感受到地磁信息,并利用地磁信息完成迁徙或归巢。地磁场信息能提供可靠导航信息,磁力线可提供罗盘信息,而磁场强度和倾角可提供位置信息。文章介绍了鸟类磁感受机制的两种重要假说——基于磁铁矿的磁感受假说和化学磁感受假说,阐明了两种假说的理论原理及实验证据,对地磁信息传导神经通路与处理脑区做了评述,并展望了其发展方向。     

The puzzle of magnetic resonance effect on the magnetic compass of migratory birds

Experiments on the effect of radio‐frequency (RF) magnetic fields on the magnetic compass orientation of migratory birds are analyzed using the theory of magnetic resonance. The results of these experiments were earlier interpreted within the radical‐pair model of magnetoreception. However, the consistent analysis shows that the amplitudes of the RF fields used are far too small to noticeably influence electron spins in organic radicals. Other possible agents that could mediate the birds' response to the RF fields are discussed, but apparently no known physical system can be responsible for this effect. Bioelectromagnetics 30:402–410, 2009. © 2009 Wiley‐Liss, Inc.     

Acuity of a Cryptochrome and Vision-Based Magnetoreception System in Birds

The magnetic compass of birds is embedded in the visual system and it has been hypothesized that the primary sensory mechanism is based on a radical pair reaction. Previous models of magnetoreception have assumed that the radical pair-forming molecules are rigidly fixed in space, and this assumption has been a major objection to the suggested hypothesis. In this article, we investigate theoretically how much disorder is permitted for the radical pair-forming, protein-based magnetic compass in the eye to remain functional. Our study shows that only one rotational degree of freedom of the radical pair-forming protein needs to be partially constrained, while the other two rotational degrees of freedom do not impact the magnetoreceptive properties of the protein. The result implies that any membrane-associated protein is sufficiently restricted in its motion to function as a radical pair-based magnetoreceptor. We relate our theoretical findings to the cryptochromes, currently considered the likeliest candidate to furnish radical pair-based magnetoreception.     

Magnetic intensity affects cryptochrome-dependent responses in Arabidopsis thaliana

Cryptochromes are blue-light absorbing photoreceptors found in many organisms where they have been involved in numerous growth, developmental, and circadian responses. In Arabidopsis thaliana, two cryptochromes, CRY1 and CRY2, mediate several blue-light-dependent responses including hypocotyl growth inhibition. Our study shows that an increase in the intensity of the ambient magnetic field from 33–44 to 500 μT enhanced growth inhibition in A. thaliana under blue light, when cryptochromes are the mediating photoreceptor, but not under red light when the mediating receptors are phytochromes, or in total darkness. Hypocotyl growth of Arabidopsis mutants lacking cryptochromes was unaffected by the increase in magnetic intensity. Additional cryptochrome-dependent responses, such as blue-light-dependent anthocyanin accumulation and blue-light-dependent degradation of CRY2 protein, were also enhanced at the higher magnetic intensity. These findings show that higher plants are sensitive to the magnetic field in responses that are linked to cryptochrome-dependent signaling pathways. Because cryptochromes form radical pairs after photoexcitation, our results can best be explained by the radical-pair model. Recent evidence indicates that the magnetic compass of birds involves a radical pair mechanism, and cryptochrome is a likely candidate for the avian magnetoreception molecule. Our findings thus suggest intriguing parallels in magnetoreception of animals and plants that appear to be based on common physical properties of photoexcited cryptochromes.     

Magnetic orientation and magnetoreception in birds and other animals

Animals use the geomagnetic field in many ways: the magnetic vector provides a compass; magnetic intensity and/or inclination play a role as a component of the navigational map, and magnetic conditions of certain regions act as sign posts or triggers, eliciting specific responses. A magnetic compass is widespread among animals, magnetic navigation is indicated e.g. in birds, marine turtles and spiny lobsters and the use of magnetic sign posts has been described for birds and marine turtles. For magnetoreception, two hypotheses are currently discussed, one proposing a chemical compass based on a radical pair mechanism, the other postulating processes involving magnetite particles. The available evidence suggests that birds use both mechanisms, with the radical pair mechanism in the right eye providing directional information and a magnetite-based mechanism in the upper beak providing information on position as component of the map. Behavioral data from other animals indicate a light-dependent compass probably based on a radical pair mechanism in amphibians and a possibly magnetite-based mechanism in mammals. Histological and electrophysiological data suggest a magnetite-based mechanism in the nasal cavities of salmonid fish. Little is known about the parts of the brain where the respective information is processed.     

Learned and spontaneous magnetosensitive behaviour in the Roborovski hamster (Phodopus roborovskii)

Sensing the geomagnetic field, called magnetoreception, might be a helpful tool for an animal to orientate and navigate in its environment. Although several rodent species are known to be magnetosensitive, detailed insights into this sensory ability are rare and the underlying mechanism in mammals is still unknown. The magnetic sense of the Djungarian hamster (Phodopus sungorus) expresses a learned behavioural pattern. Here, we report evidence for magnetoreception based on learned cues as well as spontaneous magnetosensitive behaviour in a closely related species, the Roborovski hamster (Phodopus roborovskii), for the first time. The hamsters learned to build their nests in specific magnetic directions (nest‐building assay) and spent spontaneously more time exploring a magnet compared to a sham (magnetic object assay). Furthermore, an influence of weak radio frequency magnetic fields was observed and is discussed with respect to magnetoreception mechanisms.     

Magnetoreception in plants

This article reviews phenomena of magnetoreception in plants and provides a survey of the relevant literature over the past 80 years. Plants react in a multitude of ways to geomagnetic fields—strong continuous fields as well as alternating magnetic fields. In the past, physiological investigations were pursued in a somewhat unsystematic manner and no biological advantage of any magnetoresponse is immediately obvious. As a result, most studies remain largely on a phenomenological level and are in general characterised by a lack of mechanistic insight, despite the fact that physics provides several theories that serve as paradigms for magnetoreception. Beside ferrimagnetism, which is well proved for bacterial magnetotaxis and for some cases of animal navigation, two further mechanisms for magnetoreception are currently receiving major attention: (1) the radical-pair mechanism consisting of the modulation of singlet–triplet interconversion rates of a radical pair by weak magnetic fields, and (2) the ion cyclotron resonance mechanism. The latter mechanism centres around the fact that ions should circulate in a plane perpendicular to an external magnetic field with their Lamor frequencies, which can interfere with an alternating electromagnetic field. Both mechanisms provide a theoretical framework for future model-guided investigations in the realm of plant magnetoreception.     

Angular Precision of Radical Pair Compass Magnetoreceptors

The light-dependent magnetic compass sense of night-migratory songbirds is thought to rely on magnetically sensitive chemical reactions of radical pairs in cryptochrome proteins located in the birds’ eyes. Recently, an information theory approach was developed that provides a strict lower bound on the precision with which a bird could estimate its head direction using only geomagnetic cues and a cryptochrome-based radical pair sensor. By means of this lower bound, we show here how the performance of the compass sense could be optimized by adjusting the orientation of cryptochrome molecules within photoreceptor cells, the distribution of cells around the retina, and the effects of the geomagnetic field on the photochemistry of the radical pair.     

Light effects on the multicellular magnetotactic prokaryote ‘<Emphasis Type="Italic">Candidatus</Emphasis> Magnetoglobus multicellularis’ are cancelled by radiofrequency fields: the involvement of radical pair mechanisms

‘Candidatus Magnetoglobus multicellularis’ is the most studied multicellular magnetotactic prokaryote. It presents a light-dependent photokinesis: green light decreases the translation velocity whereas red light increases it, in comparison to blue and white light. The present article shows that radio-frequency electromagnetic fields cancel the light effect on photokinesis. The frequency to cancel the light effect corresponds to the Zeeman resonance frequency (DC magnetic field of 4 Oe and radio-frequency of 11.5 MHz), indicating the involvement of a radical pair mechanism. An analysis of the orientation angle relative to the magnetic field direction shows that radio-frequency electromagnetic fields disturb the swimming orientation when the microorganisms are illuminated with red light. The analysis also shows that at low magnetic fields (1.6 Oe) the swimming orientation angles are well scattered around the magnetic field direction, showing that magnetotaxis is not efficiently in the swimming orientation to the geomagnetic field. The results do not support cryptochrome as being the responsible chromophore for the radical pair mechanism and perhaps two different chromophores are necessary to explain the radio-frequency effects.     

Electron spin polarization in photosynthesis and the mechanism of electron transfer in photosystem I. Experimental observations.

Transient electron paramagnetic resonance (EPR) methods are used to examine the spin populations of the light-induced radicals produced in spinach chloroplasts, photosystem I particles, and Chlorella pyrenoidosa. We observe both emission and enhanced absorption within the hyperfine structure of the EPR spectrum of P700+, the photooxidized reaction-center chlorophyll radical (Signal I). By using flow gradients or magnetic fields to orient the chloroplasts in the Zeeman field, we are able to influence both the magnitude and sign of the spin polarization. Identification of the polarized radical and P700+ is consistent with the effects of inhibitors, excitation light intensity and wavelength, redox potential, and fractionation of the membranes. The EPR signal of the polarized P700+ radical displays a 30% narrower line width than P700+ after spin relaxation. This suggests a magnetic interaction between P700+ and its reduced (paramagnetic) acceptor, which leads to a collapse of the P700+ hyperfine structure. Narrowing of the spectrum is evident only in the spectrum of polarized P700+, because prompt electron transfer rapidly separates the radical pair. Evidence of cross-relaxation between the adjacent radicals suggests the existence of an exchange interaction. The results indicate that polarization is produced by a radical pair mechanism between P700+ and the reduced primary acceptor of photosystem I. The orientation dependence of the spin polarization of P700+ is due to the g-tensor anisotropy of the acceptor radical to which it is exchange-coupled. The EPR spectrum of P700+ is virtually isotropic once the adjacent acceptor radical has passed the photoionized electron to a later, more remote acceptor molecule. This interpretation implies that the acceptor radical has g-tensor anisotropy significantly greater than the width of the hyperfine field on P700+ and that the acceptor is oriented with its smallest g-tensor axis along the normal to the thylakoid membranes. Both the ferredoxin-like iron-sulfur centers and the X- species observed directly by EPR at low temperatures have g-tensor anisotropy large enough to produce the observed spin polarization; however, studies on oriented chloroplasts show that the bound ferredoxin centers do not have this orientation of their g tensors. In contrast, X- is aligned with its smallest g-tensor axis predominantly normal to the plane of the thylakoid membranes. This is the same orientation predicted for the acceptor radical based on analysis of the spin polarization of P700+, and indicates that the species responsible for the anisotropy of the polarized P700+ spectrum is probably X-. The dark EPR Signal II is shown to possess anisotropic hyperfine structure (and possibly g-tensor anisotropy), which serves as a good indicator of the extent of membrane alignment.