THE STATE OF MANGANESE IN THE PHOTOSYNTHETIC APPARATUS DETERMINED BY X-RAY ABSORPTION SPECTROSCOPY

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THE STATE OF MANGANESE IN THE

PHOTOSYNTHETIC APPARATUS DETERMINED BY X-RAY ABSORPTION SPECTROSCOPY

V. Yachandra, R. Guiles, A. Mcdermott, R. Britt, J. Cole, S. Dexheimer, K.

Sauer, M. Klein

To cite this version:

V. Yachandra, R. Guiles, A. Mcdermott, R. Britt, J. Cole, et al.. THE STATE OF MAN- GANESE IN THE PHOTOSYNTHETIC APPARATUS DETERMINED BY X-RAY ABSORP- TION SPECTROSCOPY. Journal de Physique Colloques, 1986, 47 (C8), pp.C8-1121-C8-1128.

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THE STATE OF MANGANESE IN THE PHOTOSYNTHETIC APPARATUS DETERMINED BY X-RAY ABSORPTION SPECTROSCOPY

V.K. YACHANDRA, R.D. GUILES, A. McDERMOTT, R.D. BRITT, J. COLE, S.L. DEXHEIMER,

K.

a n d

Laboratory of Chemical Biodynamics, Lawrence Berkeley

Laboratory, University of California, Berkeley, CA 94720, U.S.A.

Abstract -

We present our results of X-ray edge and EXAFS studies of Mn in intermediate states (So-S4) of the photosynthetic

evolving complex prepared from spinach and the thermophilic cyanobacterium Synechococcus. We observed a shift to higher energy in the X-ray absorption K-edge energy of Mn upon advancement from the dark adapted S, state to the light-induced S, state in spinach and in Synechococcus. We have recently analysed the Mn K-edges of samples poised in the So and S, states. The K-edge inflection energy of the So samples is lower than that of samples poised in the S1 state, indicating a reduction in the effective positive charge on Mn in So relative to S1. The K-edge inflection energy of the Ss state is similar to that of the

state, suggesting an invariance in the oxidation state of Mn on advancing from the S, to the S, state. We have examined the Mn K-edge spectra of preparations depleted of essential peptides to determine the changes produced in the structure of the Mn complex. Mn EXAFS results for spinach S1 and S, samples and Synechococcus S1 all show a Mn neighbor at -2.7 d; and two N or 0 shells at -1.8 and 2.0 d; indicating a pox0 bridged Mn complex. We conclude from the edge and EXAFS studies that the light-induced S1 to S2 transition involves a change in the oxidation state of Mn with no change in the coordination of Mn in the 0,-evolving complex.

The similarity of the edges and EXAFS results from spinach and Synechococcus suggest that the basic structure of the Mn center in the 0,-evolving complex is conserved over a period of two billion years. Finally, an analysis of a composite of the S, and S2 spectra has revealed a fourth shell in the Fourier transform of the EXAFS spectra. Simulations have shown that it is compatible with an additional Mn shell at -3.3 d; or possibly second shell contributions from histidine ligands.

Introduction

The process of photosynthesis in green plants may be considered as a photoinduced chloro- phyll mediated transfer of electrons from water to C02 resulting in the formation of carbohy- drates and 0,. Strong oxidants produced by photosystem I1 (PSII) remove electrons from the 0,-evolving complex resulting in water oxidation. Photosystem I (YSI) produces powerful reduc- tants that donate electrons through a series of membrane bound proteins, one of which has been identified as an iron-sulfur protein containing at least two Fe-S clusters, to soluble ferredoxin and NADP, which are ultimately responsible for CO, reduction.

The primary focus of our work is directed toward the Mn-containing 02-evolving complex

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JOURNAL DE PHYSIQUE

&In-OEC) in PSII. The Mn-OEC is the least understood of the light-driven electron transfer protein complexes in chloroplasts, even though it has been the subject of extensive studies [I-31.

Studies of O2 evolution using a train of saturating light flashes have given rise to a model for the accumulation of oxidizing equivalents in which some intermediates, labeled So-S4 operate in a cyclic fashion [4].

hv hv h v hv

Recently established procedures for isolating subchloroplast PSII preparations [5,6] and the discovery of a light-induced multiline EPR signal 17-91 attributable to the S2 state of the Mn- OEC have facilitated the preparation of well characterized samples stabilized in the

and St states. We have used such detergent extracted but functional photosystem II particles to study the light-induced changes in the Mn oxidation state and ligand environment.

X-ray spectroscopy is element specific and hence well suited for situations where the metallo- protein cannot be isolated to purity. Few other spectroscopic techniques provide such specificity for the structure of the Mn complex in the photosynthetic apparatus. Detailed studies of K- edges can provide information about oxidation states and site symmetry and EXAFS can provide additional structural information. Such were the considerations which led to our original X-ray studies on whole chloroplasts. The EXAFS work indicated that the fraction of Mn thought to be involved in the OEC was contained in a binuclear cluster, and the edge studies indicated that the oxidation state of Mn was greater than two [10,11]. Using PSII particles we have demonstrated the direct participation of Mn in the light driven storage of oxidizing equivalents on the donor side of PSII [12].

Recently, we have been able to prepare and stabilize samples in the So and Ss states for study. In this report we present the edge spectra of samples poised in the So and S3 states, and the EXAFS results of S1 and S2 samples from spinach. Also we have prepared highly active 02-evolving PSII particles from the thermophilic cyanobacterium Synechococcus and we present the Mn K-edge spectra of the S1 and Sz states and EXAFS results of the S1 state.

Materials and Methods

Preparation of oxygen-evolving PSII sub-chloroplast membranes from spinach was accom- plished by a modification of two different Triton X-100 fractionation procedures and is described in detail in Ref. 11. Highly active 02-evolving PSII particles from the thermophilic cyanobac- terium Synechococcus were obtained by 8-octyl glucoside extraction WcDermott,

et al., unpublished results]. The samples from spinach had rates of oxygen evolution of 300-400 llmoles of 0, (mg of Ch1)-lh-I and contained -4 Mn atoms per reaction center assuming a photosynthetic unit of 250 ChVPSII. The cyanobacterium preparation yielded 0, rates of about 1000-1500 (mg of Ch1)-lh-I and contained about -3 Mn atoms per 100 ChlfPSII.

X-ray absorption edge spectra and EXAFS spectra were collected at the Stanford Synchrotron Radiation Laboratory, Stanford, CA, on wiggler beam line IV-1 and VI-2 using a Si(ll1) or a Si(400) double crystal monochromator during dedicated operation of the SPEAR storage ring, which provides 40-80 mA electron beams at 3.0 GeV. EXAFS was measured in fluorescence mode [14] using an NE104 plastic scintillation array similar to that described by Powers et al.

[IS], equipped with Cr fluorescence filters and Soller slit assembly [16]. The filter consisted of

0.25 mm thick Be with Cr electro-deposited to a thickness of 0.013 mm. Energy calibration was

.maintained by simultaneous measurement of the strong and narrow pre-edge feature of KMnO,

X-ray absorption samples were suspended in 20-50 mM MES buffers and -30% glycerol.

The samples were mounted in lucite sample holders with a sample area of 2mm x 3cm for incidence of the entire X-ray beam upon the sample material. The samples were supported on the distal side by mylar film. The size of the sample holders was so chosen that they could also be inserted into an Air Products Helitran cryostat for monitoring of the EPR spectrum of the sample. Dark adaptation, illumination, EPR and X-ray measurements were carried out directly in these cells. EPR spectra were recorded on a Varian E-109 spectrometer. Samples were run at 8 K at a microwave frequency of 9.21 GHz and with 100 KHz field modulation. During X-ray measurements, samples were suspended in a double walled kapton cryostat maintained between 170 and 190 K by a liquid N, boil-off jet.

The S1 samples were prepared by dark adaptation of the PSII particles for -2 h and then freezing the sample in liquid N2. The S2 samples were initially dark adapted and then were equilibrated at 190 K in a Varian V 6040 N M R temperature controller and illuminated with a 400 W tungsten lamp through a

cm water filter. The samples were then frozen in liquid N2.

The production of the S2 state was monitored by observing the appearance of the characteristic multiline EPR signal at 8 K.

Samples poised in the So state were prepared by treatment with N&OH followed by illumi- nation at 190 K or at 273 K in the presence of DCMU. N-OH is known to set the S-state cycle back two steps to a state called 118,191, which advances to the So state on illumination at 190 K or by illumination at 273 K in the presence of DCMU. Both these illumination protocols ensure that the PSII acceptor side is blocked between the primary and secondary acceptor site hence allowing the S-state cycle to advance only by one step. S3 samples were prepared in a manner analogous to the S2 samples. Subsequent to the generation of the S2 state, the samples were warmed to -273 K for 30 sec allowing electron transport to occur from the primary ac- ceptor to the secondary acceptor freeing the primary acceptor to receive another electron from the donor side of PSII [20,21]. The sample was then equilibrated at 235 K and illuminated for 10 min. The reduction of multiline signal and the regeneration of the primary acceptor quinone EPR signal at g=1.9 were used as the criteria for the generation of the S3 state.

Results and Discussion X-rav Absorption Edge Studies

The PSII preparations have permitted us to demonstrate that a significant change occurs in the Mn K-edge energy of PSII particles upon advancing from the S1 to the S2 state 112,221.

These observations established for the first time that Mn is directly involved in the storage of oxidizing equivalents.

1s-3d pre-edge feature is also seen in the edge spectra indicating the non-centrosymmetric environment of Mn in the complex.

We have studied the Mn K-edges of samples poised in the So and S3 states. The Mn K-edge spectra for spinach PSII samples in the So, S1 and S2 states are shown in Fig l a and in the S2 and predominantly in the Ss states are shown in Fig lb. The relative fraction of S2 was determined by monitoring the amplitude of the multiline EPR signal. The fraction in Ss was monitored by measuring the diminution of the multiline signal on illumination of a S2 sample at 235 K.

The Mn K-edge inflection of the S1 sample occurs at about 6551.3 eV, which is close to the

K-edge of Mn(III) complexes [I 11. The S2 edge inflection exihibits a shift to higher energy by

about 1 eV and occurs at -6552.5 eV which is between that observed for Mn(III) and Mn(1V)

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JOURNAL DE PHYSIQUE

complexes. The So edge inflection is observed at 6550.3 eV about 1 eV less than that of the

edge. The lower K-edge energy suggests that So is reduced relative to S1. Though we have been unable to generate a pure S3 state due to competing kinetic processes we estimate that the fraction of the S3 state in our samples is -50%. The K-edge inflection of the S3 state is similar to that observed for the S2 state as seen in Fig lb. We conclude that the oxidation state of Mn increases in the transition from So to S, and in the transition from S, to S, but is invariant on advancement from S2 to S3.

65%. 6530. 6580.

X-ray Energy (eV) X-ray Enerpp (eV)

Figure 1. The X-ray absorption K-edge spectra of Mn in the So, S,, S2 and S3 states of spinach PSII particles.

smoothed curve is drawn through the data points. The small pre-edge feature at -6543 eV is due to the 1s-3d bound state transition. a) The K-edge inflection energy for the So state is at 6550.3 eV, the S, state at 6551.3 eV and the S, state is at 6552.5 eV. The shifts from So to S, to S, indicate a progressive oxidation of Mn as the Mn-OEC steps through the S-State cycle. b) The K-edge inflection of the& state is approximately at the same position as the inflection for the S2 state, indicating that the oxidation state has not changed during the S, to S3 transition.

Recently we have prepared PSII particles from a thermophilic cyanobacterium Synechococ- cus. The particles are free of PSI and retain full 0,-evolving capacity. Using these particles we have generated a low temperature multiline EPR signal similiar to that observed in the S2 state of spinach [7]. In Fig 2 are shown the Mn K-edge spectra for Synechococcus PSII particles in the S1 and S, states. The K-edge inflection for the S, state occurs at -6551.1 eV. On advancement to the S, state the edge energy shifts to higher energy of about 6552.1 eV. The edge position and shape are similar to those observed for spinach, including the pre-edge 1s-3d bound state transition. This indicates the similarity of the M n complex in spinach and Synechococcus.

CaCl, washing of PSII particles is known to release three peptides of molecular weight 16, 24 and 33 kDa, which have been shown to be involved in 0, evolution [23]. Recently, we have extended our X-ray K-edge studies to CaC1, washed PSII particles in order to understand the structural changes produced in the Mn complex as a result of the release of the three peptides.

Incubation of PSII particles in 800 mM CaC1, buffers for 2 h releases the three peptides.

Subsequent 12 h incubation at low ionic strength additionally releases 2 Mn atoms/PSII reaction

center from the 4 W S I I that are normally present in active preparations [24]. The Mn K-edge

spectra of samples depleted of the three peptides and containing 4 W S I I and 2 MnlPSII are

shown in Fig 3. The K-edge spectrum of the 4 W S I I sample is similar to the control S1 sample,

while the 2 W S I I sample is distinctly different in shape and position with an inflection point

in spite of the loss of the three peptides and inactivation of O2 evolution. However, when 2 MnIPSII are also released a major change occurs in the structure of the Mn complex as evidenced by the dramatic change in the edge shape, the disappearance of the pre-edge feature, and the position of the inflection, which suggests a +2 oxidation state for Mn.

X-Ray Energy (eV)

Figure 2. The X-ray absorption K-edge spectra of Mn in PSII particles from the thermophilic cyanobacterium Synechococcus.

The Mn K-edge inffection for the Sl state is at 6551.1 eV and at 6552.1 eV for the S2 state. Note the pre-edge transition at -6543 eV. Smoothed curves are drawn through tke data points.

Figure 3. The Mn K-edge spectra of CaC1, washed PS II particles. Smoothed curves are drawn through the data points.

The control is a S1 sample. The 4MnPSII sample is from PSII particles from spinach incubated in CaC1, buffers for 2 h and which are depleted of the 16, 24 and 33 kDa pep- tides, while the 2Mn/PSII sample was incu- bated overnight and contains only 2MnPSII.

The inflection energy for the 4Mn/PSII sam- ple is at'6551.9 eV and at 6548.9 eV for the 2MnfPSII sample.

EXAFS Studies

The EXAFS results for the spinach PSII particles show that the salient features of the Mn structure are essentially identical for samples in the S1 and S2 states. (The greater amplitude of the first Fourier peak for the S2 sample is due to a background removal artifact.) These features are a Mn neighbor at -2.7 A, ^N or 0 ligands at -1.8 A and additional N or 0 ligands at -2.00

A. Such coordination is consistent with a d i - p - ~ ~ ~ binuclear complex 1251. The great similiarity of the EXAFS for samples prepared in S, and S, indicates that the light induced edge shift results from a change in Mn oxidation state with no change in the coordination of Mn in the O2 -evolving complex.

In Fig 4 are shown EXAFS Fourier transforms of the'Synechococcus PSII particles and from

spinach PSII particles in the

and S2 states. The three R-space features observed in spinach are

also present in Synechococcus. The parameters obtained from simulations to the fourier filtered

data from the fourier peaks labelled I, I1 and IlI in Fig 4 are shown in Table 1. These data

demonstrate that the structure of the Mn complex in the 02-evolving system is largely conserved

over the two billion year evolutionary period that separates the two species. We therefore suggest

that the bridged Mn complex is an essential feature of the 02-evolving complex.

JOURNAL DE PHYSIQUE

syneehococcus S1

;

- t

.-

- Figure 4. Fourier transforms of the klx(k) M n

-

EXAFS data from spinach S1, spinach

and

Synechococcus S,. The peaks labelled I and I1 are characteristic of bridging and terminal N or

ligands and peak I11 is due to a neighboring Mn atom and the distances are typical for

0x0 bridged binuclear Mn clusters.

Distance R + 4 ) / 2 (A)

Parameters Extracted from the Mn EXAFS Data of PS I1 Particles in the $ and S, State from Spinach and from the Thermophilic Cyanobaderium Synechococcus

Distance tA) spinach Synechococcus

S1 S2 S1

I Mn-0 or N Bridging ligand 1.75 1.76 1.85 II Mn-0 or N Terminal Ligand 2.00 1.98 2.08

Ill Mn-Mn 2.70 2.72 2.71

We estimate the uncertainty in the distances to be 0.03 A except for the bridging ligand distances where it is 0.05 A. The fits were performed using theoretical phase and amplitude functions by the Teo-Lee method [29].

There is growing concensus that there are about four M n atoms per PSII reaction center.

Our third R-space Fourier peak fits best to 1-2 Mn atoms at the distance of 2.7 A. A careful reexamination of the data shown in Fig 4 suggests that there may be an additional R-space peak beyond that now assigned to Mn. As discussed above and shown in Table I, there is virtual identity of the EXAFS for spinach PSII particles in the S, and S, states. We thus believed it reasonable to add the k-space spectra to improve the SIN. The Fourier transform of the k1 weighted sum is shown in Fig 5, which shows a fourth R-space peak significantly above the noise level. The improvement in S/N is apparent. Also shown in Fig 5 is the Fourier transform of the k2 weighted data set.

One of the procedures used to discriminate between lower-Z and higher-Z backscatterers is to

compare the FT of the data at different k-weightings. The higher Z scatterers are emphasized by

higher k-weighting because the peak in their amplitude function occurs at higher k values. The

proportional growth of both the third and fourth R-space peaks shown in Fig 5 with increased

k-weighting suggests that the fourth peak may also be assigned to Mn. If this assignment is

correct, this would imply that the four Mn atoms in the Mn-OEC are present in the form of

a tetrameric cluster. A reasonable fit is obtained to the back-transformed data from the fourth

C, N or 0 at 3.0 and 3.2 A, that would occur from binding to a histidine ring, a common metal coordinating group in proteins. Although the k-weighting behaviour of the wave is indicative of a heavy atom, one cannot unequivocally rule out the possibilty of contributions from second shell imidazole atoms.

Figure 5. Fourier transform of the sum of the S1 and Sz EXAFS data weighted by k1 and k2. The solid line is the k1 weighted data set and the dashed line is the k2 data set. Note that the third and fourth peaks grow with increasing k-weighting indicating a heavy scatterer like manganese.

Distance R + a(k)/2

(A)

Chloride ion is essential for oxygen evolution and it has been suggested that this ion might be a ligand of Mn [26,27]. We have addressed this question by both EXAFS and EPR. We have attempted to force a C1 backscattering shell into the EXAFS simulations for samples in both S1 and S2. In all cases, either the quality of fit is reduced or the number of C1 atoms included approaches zero. This result also addresses the question of sulfur coordination to Mn as the backscattering parameters of C1 and S are quite similar. We have prepared PSI1 particles in which Br- has replaced C1- with complete restoration of oxygen evolving capacity. The high resolution EPR spectra of corresponding samples again indicates that there is no statistically significant difference between them suggesting that halide does not directly coordinate Mn [28].

We are grateful to Dr. David Goodin for many helpful discussions concerning X-ray fluo- rescence detection. We thank Rick Storrs for help with data colIection. This work was supported by a grant from the National Science Foundation (PCM 82-16127 and PCM 84-16676) and by

i& Director, Office of Energy Research, Office of Basic Energy Sciences, Division of giological

Energy Conversion and Conservation of the Department of Energy under contract DE-AC03-76 SF00098. Synchrotron radiation facilities were provided by the Stanford Synchrotron Radia- tion Laboratory which is supported by the U.S. Department of Energy, Office of Basic Energy Sciences, and by the NIH Biotechnology Program, Division of Research Resources.

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