pubs.acs.org/JPCL
Basic Molecular Unit Involved in Charge Migration in Oxidized Light-Harvesting Complex 1 of Rhodobacter sphaeroides
Petersen L. Hasjim,† Friedhelm Lendzian,‡ Nina Ponomarenko,† and James R. Norris*,†,§ †
Department of Chemistry, University of Chicago, Chicago, Illinois 60637, ‡Technical University of Berlin, D-10623 Berlin, Germany, and §Institute of Biophysical Dynamics, University of Chicago, Chicago, Illinois 60637
ABSTRACT The light-harvesting complex 1(LH1) of bacterial photosynthesis harvests sunlight and transfers the energy to its inner reaction center (RC) core to drive the photosynthetic process. After chemical oxidation of the LH1 complexes of Rhodobacter sphaeroides, charge migration within the bacteriochlorophyll a (BChla) molecular array is observed at very low temperature, suggesting this protein complex as a paradigm for charge migration in the solid state and nanotechnology development. A fundamental question is whether the two BChla molecules of the R/β BChla2 structural building block function as an interacting unit for charge migration or as individual molecules. The basic charge-migration unit of LH1 of Rb. sphaeroides has been investigated by continuous-wave electron nuclear double resonance (ENDOR) at 10 K. From the hyperfine coupling constants of the unpaired electron with its neighboring atoms, the basic charge-migration unit explored by ENDOR is consistent only with a monomer of BChla. SECTION Biophysical Chemistry
were conducted by continuous-wave EPR.12,15 The broadest EPR line width of oxidized LH1 at 4 K was as high as 13 G,12 very close but not equal to the monomeric BChlaþ of 14 G.16 Functional characterization is the first step toward understanding the unusual electron transfer in oxidized LH1 at low temperature. To provide more detailed information on the free radical observed by EPR, continuous-wave ENDOR was conducted at 10 K to provide more direct information on the fundamental unit of electron spin delocalization, that is, the fundamental effective size of the observed cation radical species in LH1. Because ENDOR examines nuclei in the vicinity of unpaired electrons, this double resonance technique can provide hyperfine interactions (hfi) that CWEPR cannot, especially for cations of BChla, where EPR provides no specific hfi's. By providing discrete hfi's, ENDOR can characterize the wave function of the delocalized, unpaired electron as characterized by a spin Hamiltonian. In BChla cations, the hfi of a particular nucleus is proportional to the electron spin density in the vicinity of the nucleus. Thus, the smaller the hfi, the lower the electron density at that atom. If the hfi's observed in LH1 match the hfi's of monomeric BChla, then the fundamental cation observed in LH1 is a monomer of BChla. If the observed hfi's in LH1 are significantly smaller that those in monomeric BChla, then additional delocalization has occurred, and the fundamental unit is more than a monomer of BChla.
T
he light-harvesting complexes of photosynthetic bacteria utilize assemblies of light-absorbing bacteriochlorophyll (BChl) molecules imbedded in integral membrane protein complexes to gather light to drive the initial chemical steps of the charge separation that occurs in the photosynthetic reaction center (RC).1,2 One of the most studied species in bacterial photosynthesis is Rhodobacter sphaeroides. The shape of light-harvesting complex 1 (LH1) of Rb. sphaeroides is found to be an almost closed S-shape supermolecule, where each section of the S structure encloses a RC. The pigment array of the LH1 is constructed from pairs of helical membrane spanning proteins with two BChla's per helical pair, resulting in a total of 28 membrane-spanning helices.3 These helical pairs are often referred to as R/β BChla2 subunits.4-8 The question naturally arises as to whether strong interactions occur between the BChl molecules within these pairs. Numerous studies of chemically oxidized LH1 of Rhodobacter sphaeroides have revealed active electron/hole transfer within the system9 even at extremely low temperature.10-13 This unique temperature-dependent electron/hole-transfer property is of special interest because this protein complex constitutes a type of molecular wire as defined by Ratner et al.14 One operational issue arising regarding the electron/hole transfer within the LH1 ring is the determination of the fundamental molecular unit of charge delocalization within the LH1 complex. The dimeric form suggested by the R/β BChla2 subunits would result in a different dynamics scale for electron/hole migration in oxidized LH1 than would exist for a monomeric unit. Previous efforts to determine the fundamental charge delocalization unit of LH1
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Received Date: April 11, 2010 Accepted Date: May 10, 2010 Published on Web Date: May 12, 2010
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DOI: 10.1021/jz100471z |J. Phys. Chem. Lett. 2010, 1, 1687–1689
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Figure 2. The molecular structure and numbering scheme of BChla that designates the observed ENDOR hyperfine couplings.
At 80 K, the basic pattern of the ENDOR spectrum of oxidized LH1 is consistent with a monomer of BChla.13 Although the EPR line width at 80 K decreased as the extent of oxidation increased, the ENDOR pattern remained constant, and only its intensity decreased with increased oxidation, leading to the conclusion that LH1 contains two types of sites.13 Some sites are nonmigrating deep traps for the cation that are observed by ENDOR as monomer cations of BChl. Other sites of oxidation are not deeply trapped and contain the migrating spins responsible for the line narrowing observed by EPR but whose dynamics prevents observation by ENDOR.13 However, the possibility exists that at 80 K, the charge-migration process involving the fundamental unit remains hidden by thermal activation. To investigate the potential effect of thermal activation on the nature of charge migration in the fundamental unit, the ENDOR of LH1 should be examined not only as a function of the extent of oxidation but also as a function of temperature. To eliminate thermal modifications of the fundamental unit with regard to charge migration as much as possible, ENDOR must be recorded at the lowest possible temperature. Thus, to decrease thermal effects on the ENDOR spectrum and the corresponding fundamental charge-migration unit of oxidized LH1, we report ENDOR that has been obtained at 10 K. The ENDOR experiment was conducted at 10 and 80 K on a mildly 2.7% oxidized LH1. The ENDOR spectra are shown in Figure 1. The corresponding CWEPR line widths are about 12 and 10 G for 10 and 80 K, respectively. The EPR line width of the 10 K system is relatively close to the monomeric BChla radical's line width. The low-temperature ENDOR spectra show heavy distortion of the ENDOR signals due to anisotropy induced by the frozen state at cryogenic temperatures. Both spectra, despite the significant difference in CWEPR line width, feature several peaks with a symmetry axis in the middle noted by the presence of a sharp matrix line. This type of spectrum was observed before for ENDOR16 and special TRIPLE17 resonance measurements of monomer BChlaþ in
the frozen state. The frozen-state spectra contrast with the liquid-state measurement obtained in a 6 to 1 mixture of CH2Cl2 and CH3OH, where the spectrum exhibited many first derivative peaks that were due to the coupling of protons, methyls, and methines to the unpaired electron.17 Because the detected hyperfine interactions in the LH1's ENDOR spectrum exhibit almost axial symmetry, the coupling constants could be determined based on the value of the parallel and perpendicular components of the coupling constants. The molecular structure of BChla along with the numbering scheme used for the coupling constants is shown in Figure 2. The positions of the strongest peaks, each pair about 2 and 4.5 MHz away from the matrix line, are the same as those detected for the methyl #5 and #6 in monomeric BChlaþ's TRIPLE/ ENDOR spectrum in the frozen state. From the 10 K ENDOR spectrum, the parallel and perpendicular components of the coupling constants corresponding to methyl #5 and #6 are A^(#5) =4.2 MHz, A (#5) =5.6 MHz and A^(#6) =9.0 MHz, A (#6)=10.8 MHz, respectively. The estimated coupling constant is given by A=1/3(A þ 2A^), yielding A(#5)=4.67 MHz and A(#6)=9.6 MHz. These coupling constants are very comparable to those of the methyl groups in the monomeric BChla radical of 4.93 MHz (methyl #5) and 9.62 MHz (methyl # 6).16,17 The coupling constants for other hydrogens and methines are not detected clearly in LH1 because at both low temperatures, those static hydrogen atoms lose their isotropy, and their signals broaden significantly and become weak in intensity. The β-H's should not lose much of their isotropy, but they are sensitive to temperature-dependent conformational changes of rings X and Y (see Figure 2) and, as a result, broaden. From the ENDOR spectra, no evidence exists that the BChla's in the LH1 are in the form of a dimer similar to the reaction center primary donor. The ENDOR spectrum of the special pair of the reaction center protein has all of the hyperfine couplings greatly reduced, reflecting delocalization of the unpaired electron over two macrocycles instead of one.18
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Figure 1. First derivative ENDOR spectra of LH1 at 10 and 80 K.
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DOI: 10.1021/jz100471z |J. Phys. Chem. Lett. 2010, 1, 1687–1689
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REFERENCES
Should that be the case for LH1 at 10 K, the appearance of the strong 2.3 MHz peak would be observed due to the shift of the methyl #5's 4.9 MHz coupling constant in the monomeric BChlaþ ENDOR, while the 9.6 MHz coupling constant that belongs to methyl #6 would shift to around 4.8 MHz. As a result, there should be no 9.6 MHz coupling constant observed in the ENDOR spectrum. On the basis of the observed coupling constants, the ENDOR spectrum of LH1 resembles a monomeric BChla spectrum instead of a dimeric one as in the RC. However, unlike room-temperature ENDOR of monomeric BChlaþ, the BChla's in LH1 samples were frozen so that many weaker couplings broadened unclearly. In short, no evidence exists for a dimer cation at low temperature such that from the functional perspective, the ENDOR spectra at 10 and 80 K support monomeric BChla as the basic unit that constitutes the backbone of LH1.
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EXPERIMENTAL SECTION
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The LH1 protein complex was prepared from the Rb. sphaeroides puc705-BA strain. The bacterial culture and isolation of the LH1 complex was conducted by following a procedure from previous work.10-12 The final detergent-isolated LH1 was suspended in 10 mM Tris HCl buffer (pH 7.9, 0.8% BOG, 10 μM EDTA). Radical cations of the LH1 complexes were generated by oxidation with a solution containing both potassium ferricyanide, K3Fe(CN)6, and potassium ferrocyanide, K4Fe(CN)6, under ambient conditions. Stock solutions of the oxidizing reagent were prepared in 10 mM Tris HCl buffer (pH 7.9, 0.8% BOG, 10 μM EDTA) and used immediately. Optical absorbance spectra for the LH1 complexes were measured under ambient conditions with a Shimadzu UV-1601 spectrophotometer. Quartz cuvettes of 1 mm path length were used for all optical measurements. The fraction of oxidized BChla, χ, was determined by the bleaching of the near-IR band characteristic of the BChla bound in the LH1 complex. Specifically, the optical absorbance spectrum at 880 nm was measured before and after chemical oxidation of the complexes, giving the fraction oxidized as χ = [(Abefore - Aafter)/Abefore] = (ΔA/Abefore). The absorbance after oxidization was corrected for the decrease due to dilution. The fraction oxidized for the LH1 in this sample was 0.027 and is referred to as 2.7% oxidized LH1. The CW-ENDOR spectrum was measured on a Bruker ESP 300E spectrometer using a self-built ENDOR accessory, which consists of a Rhode & Schwarz RF synthesizer (SMT02), an ENI A200L solid-state RF amplifier, and a self-built high-Q TM110 ENDOR cavity. To obtain the ENDOR spectra, the oxidized LH1 sample was saturated at the center of the EPR resonance with 10 mW microwave power, and 100 Wof radio frequency power was applied.
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AUTHOR INFORMATION Corresponding Author:
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*To whom correspondence should be addressed. E-mail: jrnorris@ uchicago.edu.
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ACKNOWLEDGMENT Support from the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Contract DE-FG02-96ER14675 is gratefully acknowledged.
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