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Copyright © 2005 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 89, Issue 4, L28-L30, 1 October 2005

doi:10.1529/biophysj.105.069609

Biophysical Letters

An Anomalous Distance Dependence of Intraprotein Chlorophyll-Carotenoid Triplet Energy Transfer

Hanyoup Kim^*, 1, Naranbaatar Dashdorj^*, 1, Huamin Zhang^†, Jiusheng Yan^†, William A. Cramer^† and Sergei Savikhin^*, ,  

^* Department of Physics, Purdue University, West Lafayette, IN 47907
^† Department of Biological Sciences, Purdue University, West Lafayette, IN 47907

Address reprint requests and inquiries to Sergei Savikhin .

¹ Hanyoup Kim and Naranbaatar Dashdorj contributed equally to this work.

The membrane-bound cytochrome b₆f complex in oxygenic photosynthesis (Figure 1A) mediates electron transfer between the reaction centers of photosystems I and II and facilitates coupled proton translocation across the membrane. It contains a single chlorophyll (Chl) a molecule that is known to produce highly toxic singlet oxygen (¹O₂) as the result of energy transfer from its excited triplet state to the oxygen molecule 1. The recent x-ray structures of the b₆f complex show that the β-carotene is 14Å from the Chl a2,3—too far for protection against singlet oxygen formation via the conventional mechanism of direct quenching of the Chl a triplet excited state by β-carotene 4. We have recently reported that, unlike other known Chl-containing protein complexes, the formation of singlet oxygen by the Chl a in the b₆f complex is reduced by a factor of ∼25 by the unusually short singlet excited state lifetime of the Chl a5. The time-resolved optical experiments reported in the current work reveal that additional protection in the cytochrome b₆f complex is provided by rapid triplet-triplet excitation energy flow between the Chl a and β-carotene that unexpectedly occurs over the large distance, and must involve an unconventional mechanism.To examine triplet-triplet energy transfer in the cytochrome b₆f complex, the dynamics of absorption changes associated with the excited states of the Chl a and β-carotene were probed. Samples of conventionally purified cytochrome b₆f complex, as well as of complex that was further refined to an ultra-pure state by dissolving diffraction-quality crystals, were excited within the Chl a absorption band at 660 nm and probed at 520 nm and 670 nm under aerobic conditions (Fig. 2). The 520 nm kinetic profile in both samples features a major exponential component with a decay time of 2.6±6 0.5μs (Figure 2AB). The decayassociated spectrum of the 2.6μs component formed a distinct band centered at 520 nm (Figure 2C). The lifetime and spectral shape of this component are consistent with the triplet-singlet spectrum of a carotenoid molecule, which has been shown to have a lifetime of 2–4μs under aerobic conditions 6. The excitation spectrum of the 2.6μs component mimicked the Qy absorption band of the Chl a centered at 670 nm, indicating that the triplet excited state of carotenoid (³Car*) is created as the result of the Chl a excitation. No signal associated with ³Chl* was resolved in the complex purified through crystallization (Figure 2A, 670 nm profile). It is inferred that the characteristic time for triplet energy transfer from the Chl a that is integral to the cytochrome b₆f complex to the β-carotene is shorter than the time resolution (∼8ns) of the instrument, so that the signal escapes detection. For conventionally purified complex, that was not further purified by crystallization, and which thereby contains a small amount (∼20% of the total Chl a) of adventitiously bound Chl, the photobleached signal probed at 670 nm decays with a lifetime of ∼110 ns (Figure 2D). This is consistent with the expected lifetime of the triplet excited state of a monomeric Chl (³Chl*) under aerobic conditions, where the chlorophyll triplet energy is transferred to the triplet-singlet transition of molecular oxygen (1) and indicates that this signal originates entirely from Chl onspecifically bound to the b₆f complex.

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Figure 1

(A) Lumenal side view of the dimeric cytochrome b₆f complex 2. (B) A cross-section (black) cut through the structure reveals a cavity formed by hydrophobic residues that may serve as an oxygen channel for shuttling triplet excitation energy from the Chl a (green) to β-carotene (orange). Five hydrophobic residues (Ile, Leu, Phe, Met, and Val) are proposed to form an oxygen channel (blue) 9,10.

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Figure 2

(A) Time-resolved transient absorption difference profiles probed at 520nm and 670nm, which represent triplet ³Car* and ³Chl* population dynamics. The b₆f complex purified via crystallization was excited at 660nm under aerobic conditions. The amplitude of the absorbance changes at 670nm is zero relative to the noise level. (B) Transient absorption measured at 520nm for the conventionally purified complex under the same conditions. (C) The amplitude of the 2.6μs component associated with ³Car* formation shown in panel B as a function of probe wavelength. (D) Transient absorption signal probed at 670nm for the conventionally purified complex is not zero and stems from nonspecifically bound chlorophylls.

These experimental results unambiguously demonstrate that a substantial amount of ³Car* is formed from excitation of the Chl a in the cytochrome b₆f complex, and imply that an effective triplet-triplet energy transfer channel exists between the Chl a and β-carotene. No signal associated with the formation of the integral ³Chl*, nor rise time in ³Car* population (Figure 2AB), was resolved in these experiments, which sets the upper limit for this triplet-triplet energy transfer to <8ns (the time resolution of our experimental setup).

Using the theory of Dexter 7, we estimated that the rate of the direct triplet-triplet energy transfer from the ³Chl* to β-carotene in the cytochrome b₆f complex should be ∼(0.3ms)⁻¹, which is ∼5 orders of magnitude slower than the upper limit of the ³Car* formation time observed in the b₆f complex. Thus, not surprisingly, the conventional mechanism of singlet oxygen protection by direct triplet-triplet energy transfer process between the Chl and Car separated by 14Å does not function in the cytochrome b₆f complex.

It is proposed that oxygen mediates triplet energy transfer between the Chl a and β-carotene. Oxygen can effectively accept triplet-excited state energy from ³Chl*, forming singlet oxygen 1, and it is well known that singlet oxygen in solvents can be effectively quenched by a carotenoid, promoting the latter into the triplet excited state 4,8.

To facilitate rapid energy transfer, oxygen could be confined in its diffusive motion to an intraprotein channel connecting the Chl a and Car, causing a significant increase in the local oxygen concentration and the rate of the oxygen-mediated Chl a triplet state quenching. An oxygen channel has been described that facilitates oxygen transfer within cytochrome c oxidase, which catalyzes the reduction of oxygen to water 9. Simulations of this process by molecular dynamics 10 show that molecular oxygen shuttles along a single well-defined ∼15Å long pathway in a time on the order of tens of picoseconds, with a low probability of escape from this channel. The involvement of mobile oxygen in the triplet-triplet energy transfer in the cytochrome b₆f complex isolated from a cyanobacterium would be consistent with the absence of the ³Car* signal at 77K reported by Peterman et al. 11—the mobility of oxygen would be greatly impeded at low temperatures. We have confirmed this result (data not shown).

Both experimental studies and molecular dynamic simulations imply that an effective intraprotein oxygen channel could be formed by hydrophobic residues 9,10. Structural analysis of the cytochrome b₆f complex reveals that there is, indeed, an open pathway surrounded primarily by hydrophobic residues (Figure 1B). Since the rate of the ¹O₂ quenching by β-carotene is more than two orders of magnitude greater than the reactivity of ¹O₂ toward the surrounding amino acids 12, this mechanism for triplet energy transfer to β-carotene would allow it to serve a protective function. The reason for distant placement of the necessary protective β-carotene relative to chlorophyll a remains a question.

Purification and crystallization of the cytochrome b₆f complex from Mastigocladus laminosus is described in detail elsewhere 13. Conventionally purified complexes (i.e., purified without crystallization) contained ∼1.2 Chl a molecules per cytochrome f. Control experiments were performed on x-ray diffraction quality single crystals of the cytochrome b₆f complex dissolved in a buffer. These samples had a stoichiometry of Chl a 1.0:1 relative to cytochrome f. All complexes were in a functionally active form.

Transient absorption difference measurements were carried out by laser flash photolysis using alternatively ∼20ns or 100 fs full width at half-maximum excitation pulses at ∼660nm. The time resolution was limited only by the light detectors (∼8ns).