Ferredoxin and £Avodoxin Reduction by Photosystem I

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Biochimica et Biophysica Acta 1507 (2001) 161^179 www.bba-direct.com

Review Ferredoxin and £avodoxin reduction by photosystem I

Pierre Se¨tif *

Section de Bioe¨nerge¨tique and CNRS URA 2096, De¨partement de Biologie Cellulaire et Mole¨culaire, CEA Saclay, 91191 Gif sur Yvette, France

Received 11 December 2000; received in revised form 20 April 2001; accepted 1 May 2001

Abstract

Ferredoxin and flavodoxin are soluble proteins which are reduced by the terminal electron acceptors of photosystem I. The kinetics of ferredoxin (flavodoxin) photoreduction are discussed in detail, together with the last steps of intramolecular photosystem I electron transfer which precede ferredoxin (flavodoxin) reduction. The present knowledge concerning the photosystem I docking site for ferredoxin and flavodoxin is described in the second part of the review. ß 2001 Elsevier Science B.V. All rights reserved.

Keywords: Photosystem I; Ferredoxin; Flavodoxin; Electron transfer; Iron^sulfur cluster; Docking site

1. Introduction to the oxidation of a chlorophyll a dimer, named P700, and to the reduction of one of the two terminal Photosystem I (PSI) is found in all types of oxy- acceptors FA or FB, which are [4Fe^4S] clusters. genic photosynthetic organisms and operates as a Plastocyanin or cytochrome c6 are oxidized by light-driven oxidoreductase which transports elec- P700 on the lumenal side of PSI whereas ferredoxin 3 trons from one face of the photosynthetic membrane (Fd) or £avodoxin (Fld) are reduced by (FA,FB) on to the other. It is a multisubunit protein complex the stromal side of PSI. Fd and Fld are involved in consisting of v11 subunits (Xu et al., this issue; multiple redox reactions. They can both sustain cy- Scheller et al., this issue) and contains many cofac- clic as well as linear electron £ow. The cyclic electron tors, with light-capture or electron transfer (ET) £ow creates a proton gradient across the photosyn- roles. Light absorption leads to a charge separation thetic membrane and allows ATP synthesis inde- forming a primary radical pair which is stabilized by pendent of photosystem II activity. During linear successive ET steps (Brettel and Leibl, this issue). electron £ow, reduced Fd (Fld) provides the elec- These intramolecular ET reactions lead ultimately trons necessary for NADP reduction in a reaction catalyzed by ferredoxin:NADP-oxidoreductase (FNR). Water dissociation by photosystem II is the source of electrons during linear electron £ow. Fd Abbreviations: Em, midpoint redox potential; ET, electron serves also as an electron donor to a number of transfer; FA,FB,FX, the three [4Fe^4S] clusters of photosystem soluble enzymes involved in nitrogen metabolism, I; Fd, ferredoxin; Fld, £avodoxin; FNR, ferredoxin:NADP- reductase; PSI, photosystem I; WT, wild-type sulfur metabolism and the regulation of carbon me- * Fax: +33-1-6908-8717. tabolism [1]. Fld can substitute Fd under conditions E-mail address: [email protected] (P. Se¨tif). of iron deprivation in most cyanobacteria and some

BBABIO 45079 12-10-01 Cyaan Magenta Geel Zwart 162 P. Se¨tif / Biochimica et Biophysica Acta 1507 (2001) 161^179 algae [2,3] and may be e¤cient in reducing most or are undoubtedly relevant from a physiological point all of these soluble enzymes. of view, they may be rather complicated to interpret The PSI complex from the cyanobacterium Syn- because they involve many individual steps. In con- echococcus elongatus has been crystallized in its tri- sequence, the rate of the whole process will be lim- meric form. At the moment, data are available at a 4 ited by the slowest reaction, which may vary from Aî resolution [4] and an improved model of the sub- one experiment(alist) to another. In the absence of units PsaC, PsaD and PsaE has recently appeared any exogenous electron donor or acceptor, the for- [5]. These three subunits constitute a stromal ridge mation of the charge-separated state (P700 -(FA, 3 and were recently shown to be all involved in the FB) ) is followed by a charge recombination process interaction with Fd and Fld, as it will be detailed occurring in the 10^100 ms time range [10]. It has in the present review. Many structures of the plant- long been known that this recombination is inhibited type [2Fe^2S] Fd are available, both from X-ray ([6] if one adds methylviologen (MV) as an exogenous and references therein) and nuclear magnetic reso- electron acceptor, and this results in a slower decay nance (NMR) ([7] and references therein). A few of P700 (see, e.g., [11]). The same type of experi- X-ray structures of the photosynthetic type of Fld ment can be performed with Fd as an electron ac- were also determined ([8] and references therein). ceptor. Though these measurements are relatively There are only minor structural variations among easy to perform by monitoring P700 by £ash ab- these Fd and Fld structures. Though rather di¡erent sorption spectroscopy or by EPR, there was, to my in sizes (W11 kDa for Fds and 18^20 kDa for Flds), knowledge, no report of such data until 1993 [12,13]. both types of proteins are strongly acidic, whereas Similar observations can be made by measuring pho- the stromal surface of PSI is thought to be positively tovoltage signals which monitor membrane potential charged. Therefore electrostatic forces are of major decay kinetics either in the absence or the presence of importance for the interactions between PSI and Fd native or arti¢cial electron acceptors [14]. (or Fld). This topic will appear from time to time in In order to monitor directly the kinetics of Fd the present review but will not be the subject of a reduction, one must record absorption changes at a speci¢c discussion as it awaits a better resolved PSI wavelength where the [2Fe^2S] cluster of Fd is con- structure. I will address more particularly the mea- tributing. This was made in vitro by £ash-absorption surements of the kinetics of Fd and Fld photoreduc- spectroscopy in the visible region, either at 480 nm tion by PSI, with £ash-absorption spectroscopy as a [15,16] or in the 460^600 nm region [17,18]. These major tool of investigation. The relative contribu- measurements take advantage of the fact that reductions of the three stromal subunits to Fd (Fld) bind- tion of the [2Fe^2S] cluster of Fd leads to a larger ing and photoreduction will be also discussed togeth- bleaching than single reduction of the terminal [4Fe^ er with the identi¢cation of a few residues essential 4S] clusters FA and FB of PSI (Fig. 1). A relatively for fast ET. slow process of Fd reduction (k 6 280 s31 i.e.,

t1=2 s 2.5 ms) has been claimed to correspond to Fd reduction in [15,16]. This is in complete discrep- 2. Electron transfer from PSI to ferredoxin ancy with the fast Fd reduction that have been measured in kinetic studies made by myself and col- 2.1. In vitro measurements of the kinetics of leagues, with ¢rst-order halftimes in the Fd reduction submicrosecond and microsecond ranges and halftimes corresponding to second-order processes which Though it has long been established that Fd can be can be observed below a few hundreds of microsec- reduced by PSI, only NADP photoreduction was onds (see, e.g., [18,19]). This discrepancy cannot be used as a functional test of this enzymatic activity ascribed to the use of biological materials from dif- until relatively recently (see, e.g., [9] for a review). ferent organisms as fast kinetics can be observed in This test necessitates FNR for the ¢nal reduction any type of PSI/Fd system studied yet. step and rates of electron £ow are determined under The kinetics of Fd reduction by PSI were studied continuous illumination. Though such measurements in detail in vitro by £ash-absorption spectroscopy

BBABIO 45079 12-10-01 Cyaan Magenta Geel Zwart P. Se¨tif / Biochimica et Biophysica Acta 1507 (2001) 161^179 163

with both partners from the cyanobacterium Syn- echocystis 6803 [17,18] and from the green alga Chla- mydomonas reinhardtii [20] (Fig. 1). In both cases, it was possible to identify several fast kinetic components (submicrosecond and microsecond), the rates of which do not depend upon the concentrations of the partners, as well as a slow kinetic component, the rate of which depends linearly on the Fd concentration. In the following, these components will be re- ferred to as ¢rst-order and second-order phases, respectively. The ¢rst-order phases are thought to correspond to ET processes which occur within PSI/Fd complexes which are preformed before £ash excitation. The second-order phase (rate constant 0 kon) corresponds to a di¡usion-limited ET which is observable in the fraction of PSI which does not bind Fd before £ash excitation. The amplitudes of the ¢rst-order phases increase with the Fd concentration, which allows to calculate the dissociation constant Kd of the PSI/Fd complex. The di¡erent observable 0 parameters Kd, kon and t1=2 are shown in the scheme of Fig. 2. From this scheme, it appears that only a small subset of the parameters which are relevant for a full description of Fd reduction is available from the present data. Table 1 exhibits numerical values of 0 the parameters Kd, kon and t1=2 for Synechocystis 6803, C. reinhardtii and spinach. In the following, di¡erent aspects of Fd reduction will be discussed in detail, by raising di¡erent issues and pointing still open questions.

2.2. The [4Fe^4S] cluster FB is the distal cluster and is the partner of Fd

The ¢rst crystallographic structure of PSI revealed the asymmetrical distribution of the terminal electron acceptors, i.e., the [4Fe^4S] clusters FA and FB which are bound to the stromal subunit PsaC [21]. This Fig. 1. Spectrum and kinetics of Fd reduction. (Upper part) asymmetry is superimposed on a global 2-fold pseu- Continuous line, oxidized minus reduced di¡erence spectrum of 3 dosymmetry of the membrane part with a C axis Fd from Synechocystis 6803; full circles, ((FA,FB) 3(FA,FB)) 2 di¡erence spectrum; open squares, di¡erence of the two preced- perpendicular to the membrane plane. It was also ing spectra, corresponding to the spectrum that is expected for later recognized that the core of PsaC exhibits a local Fd reduction from (F ,F)3. (Middle and lower part) Ki- A B pseudo-C2 axis which is oriented at 62³ to the mem- netics, recorded in vitro at 580 nm, corresponding to reduction 3 brane normal [4]. Though the cysteine ligands of of Fd by (FA,FB) in Synechocystis 6803 ([PSI] = 0.18 WM; [Fd] = 1.0 WM) and Chlamydomonas reinhardtii ([PSI] = 0.14 WM, both clusters were identi¢ed (Vassiliev et al., this [Fd] = 17 WM), respectively. issue), this local symmetry impeded, until recently, to conclude from the X-ray data which one among the two clusters is proximal or distal to the mem-

BBABIO 45079 12-10-01 Cyaan Magenta Geel Zwart 164 P. Se¨tif / Biochimica et Biophysica Acta 1507 (2001) 161^179

tence of a high a¤nity complex in the absence of FB allowed to derive a value of 6 s31 for the ¢rst-order 3 ET rate from FA to Fd.

2.3. Multiplicity of ¢rst-order components of Fd reduction

At least two or three ¢rst-order components were necessary to describe satisfactorily the ¢rst-order kinetics of ferredoxin reduction in any wild-type (WT) system that was studied yet (see Table 1). A submicrosecond phase was always detected together with either one or two microsecond components. Though signi¢cantly di¡erent one from another, the spectra

of the three phases (t1=2W0.5, 15 and 100 Ws) obtained in Synechocystis 6803 were shown to be con- 3 sistent with Fd reduction from (FA,FB) [18]. From a careful examination of the kinetics observed with various preparations of PSI and Fd, the possibility that the complex kinetic pattern could arise from the partial degradation of one or both partners of the complex appeared very unlikely [17]. The relative proportions of the ¢rst-order components do not depend on the Fd concentration. This was interpreted by assuming that the di¡erent components correspond to di¡erent types of PSI/Fd complex which are mutually exclusive. As Fd reduction can occur

with t1=2 6 1 Ws in some fraction of the complexes, one obvious issue is why the process is much slower in the other fraction: there must be some rate limitation either in ET from FB to Fd or during intramolecular PSI ET. Before discussing in more detail the possibility of a rate limitation within PSI, I will Fig. 2. Kinetic scheme of Fd reduction. The observable parame- ¢rst summarize the current experimental knowledge ters in £ash-absorption experiments are shown in bold (K , k0 , d on about the terminal steps of charge separation within t ). PSI3, PSI with reduced terminal acceptors. 1=2 PSI and the intramolecular ET in some bacterial ferredoxins. brane. After some period of debate, it appears from the more recent functional results that FA and FB are 2.4. Intramolecular ET between [4Fe^4S] clusters in the proximal and distal clusters, respectively (Vassi- PSI liev et al., this issue). This is now unambiguously con¢rmed by the latest X-ray structure with a better This topic has been reviewed recently by Brettel resolution (P. Jordan, P. Fromme, N. Krauss, per- [10], at a time where most of the experimental evi- sonal communication). After selective destruction of dence was favoring FB as the proximal cluster. This FB, lack of e¤cient ET to Fd and Fld has been has been established now to be wrong and all avail- evidenced [14,22,23]. It was also found that destruc- able data can be (re)interpreted within a model where tion of FB leads to only a 3^4-fold decrease in a¤nity ET occurs along the pathway: FXCFACFB. I will of Fd for PSI in Synechocystis 6803 [22]. The exis- presently focus on the available kinetic data concern-

BBABIO 45079 12-10-01 Cyaan Magenta Geel Zwart P. Se¨tif / Biochimica et Biophysica Acta 1507 (2001) 161^179 165

Table 1 Kinetic characteristics of Fd reduction with PSI and Fd from di¡erent origins First-order phases Second-order phase k (U108 Ms)

Origin of PSI/origin of Fd Kd (WM) No. of phases t1=2 of phases (Ws) Type of PSI/type of Fd % of phases at 580 nm Synech. 6803/Synech. 6803 0.2^0.6 3 0.5/(13^20)/(85^120) 3.5 WT/WT 40/40/20 Spinach/spinach 6 32 6 1/(4^6) n.d. WT/WT 55/45 Chlam. reinh./Chlam. reinh. 6^9 2 6 1/(3^6) 2.3 WT/WT 35/65 Chlam. reinh./Synech. 6803 6 2 6 1/(9^13) 3.5 WT/WT 30/70 Synech. 6803/Synech. 6803 13 2 6 1/ 6 25 0.27 (PsaD-minus)/WT s 80/ 6 20 Synech. 6803/Synech. 6803 52 2 6 1/10 1.5 (PsaE-minus)/WT 60/40 Synech. 6803/Synech. 6803 56 2 6 1/25 0.84

R39Q(PsaE)/WT 60/40 Chlam. reinh./Chlam. reinh. 0.12 1 (2) 6 1/(6^10) n.d.

(I12V/T15K/Q16R)(PsaC)/WT 90/10 Data are given for wild-type systems (WT), for PSI without PsaD or PsaE, and for two site-directed mutants of either PsaE and

PsaC. The measurements were made at pH 8.0 and in the presence of 30 mM NaCl and 5 mM MgCl2 (data from [18^20,37,65,86]). Synech., Synechocystis; Chlam. reinh., Chlamydomonas reinhardtii; n.d., not determined.

ing forward ET at room temperature. After photo- ing the kinetics of ET between FA and FB. From excitation of PSI, the quinone(s) A1 is reduced in the [22], there was no indication of a photovoltage signal 3 picosecond time range, and A1 is reoxidized by FX associated with this step in the submicrosecond time with t1=29200 ns, as recorded by £ash-absorption, range. In another measurement made with a 1 Ws EPR and photovoltage studies (Brettel and Leibl, instrument response time and a slow dielectric relax- this issue). The measurement of photovoltage signals ation, a voltage increase was reported with time con- was the main technique for studying the following stants of 30 and 200 Ws [26]. The 30 Ws phase was steps, i.e., ET between FX,FA and FB, as these re- ascribed to ET from the proximal to the distal cluster actions are expected to be electrogenic. Kinetics of (FACFB) (see, however, Section 2 below arguing for forward ET have been measured with a nanosecond a submicrosecond reduction of FB). A single photo- time resolution with PSI membranes either in the voltage study was performed in the presence of Fd presence or in the absence of (FA,FB) [24], as well and Fld and no additional electrogenesis could be as after selective destruction of FB [22]. Both studies associated with the addition of these external accep- indicate that the proximal cluster (FA) is reduced tors, thus suggesting that reduction of either Fd or 3 3 from FX faster than FX is reduced from A1 .An Fld is electrically silent [14]. upper time constant of 50 ns was estimated for ET 3 from FX to FA (see discussion of technical limita- 2.5. Intramolecular ET between [4Fe^4S] clusters in tions in [10]). The amplitude of the photoelectric sig- bacterial ferredoxins nal attributable to this ET step appeared to be com- 3 patible with complete reduction of FA from FX. The PsaC subunit of PSI, which harbors FA and Nanosecond reduction of FA is in accordance with FB, bears a close resemblance to bacterial ferredoxins measurements made with a 200 ns time resolution containing two [4Fe^4S] clusters, especially with re- and a slower dielectric relaxation, which did not al- gard to ligation, geometry of the clusters and dis- low to resolve the ET kinetics between FX and (FA, tance between the clusters. Intramolecular ET has FB) [25]. Very little is known at the moment concern- been studied by NMR in two such bacterial ferredox-

BBABIO 45079 12-10-01 Cyaan Magenta Geel Zwart 166 P. Se¨tif / Biochimica et Biophysica Acta 1507 (2001) 161^179 ins, from Clostridium acidurici and Clostridium pas- mined at room temperature for the ¢rst and second teurianum [27]. These studies made use of the broad- reduction of (FA,FB), respectively [31]. The origin of ening of coalesced lines which arise under conditions the discrepancy between low and room temperature of intermediate reduction due to intramolecular ET determinations is still not clari¢ed. Typical Emsof 6 in the fast exchange regime. Rates of about 5U10 plant-type Fds are about 3410 mV [32] and Em of s31 ( = sum of rates from one cluster to the other and Synechocystis 6803 Fd has been measured at 3412 vice versa) have been found for both WT Fds and mV [33]. from several site-directed mutants of the C. pasteur- It was assumed in [17,18] that the ¢rst-order re- ianum Fd. Other studies made with site-directed mu- duction of Fd is almost complete at saturating Fd tants of a similar Fd from Chromatium vinosum are concentrations. In [18], the spectrum of the sum of giving rates of 0.2 to 4.0U106 s31 (rates from one the amplitudes of the absorption changes ascribed to cluster to the other; t1=2 = 0.17 to 3.5 Ws) for cases ¢rst-order phases of Fd reduction has been measured where the two clusters have similar oxidation-reduc- for Synechocystis 6803 at pH 8.0. This spectrum tion midpoint potentials (Em), i.e., with MvEmM 6 80 matches very closely both in shape and amplitude mV [90]. NMR studies have also been performed the spectrum that can be calculated for complete 3 with subunit PsaC from the cyanobacterium S. elon- reduction of Fd from (FA,FB) (see [18] for the gatus [28] and Synechococcus sp. PCC 7002 [29], but absorption coe¤cients of P700 and Fd which were no intramolecular ET rate was derived, except for a used for this calculation). Moreover, one can note lower limit (k s 104 s31). When considering the that one expects almost no change due to Fd reduc- above data about ET in bacterial ferredoxins, and tion at 460 nm (Fig. 1). This ¢ts very well with the the very close similarity between these ferredoxins reported data [18]. This observation, together with and PsaC, one cannot exclude that ET from FA to the similarity in spectral shapes between the mea- FB can occur in the microsecond time domain, espe- sured and calculated spectra, indicates that the errors cially if one has Em(FB) 6 Em(FA) (see below). in the absorption coe¤cients of P700 and Fd cannot be large. As a consequence, one can assume as a 2.6. Changes in Gibbs energy associated with ET very conservative statement that at least 80% of Fd is between PSI clusters and from (FA,FB)toFd reduced within the Synechocystis PSI/Fd complex at pH 8. This is clearly compatible with the estimate of Apart from the case of the ET step involving the Ems made by EPR for (FA,FB), from which almost quinone(s) A1 and FX, for which the existence of a 100% reduction of Fd is expected but is not with the fast preequilibrium is still a matter of debate (Brettel room temperature determination in [31], for which and Leibl, this issue), the two other ET steps leading about 60% of Fd reduction can be calculated if one to reduction of FA (A0CA1 and FXCFA) are gen- assumes no shift of FA,FB and Fd Ems due to com- erally considered to be almost complete, i.e., involv- plex formation. It should be mentioned also that the ing 100% of ET. From the comparison of the recom- available data are much less extended in the case of 3 bination reactions within the pairs (P700 ^FX ) and C. reinhardtii, as measurements were only performed 3 (P700 ^FA ), it was thus estimated that the equilibri- at 580 nm, so that one cannot give a reliable value of 3 3 um constant between (FXFA) and (FXFA) is about the amount of photoreduced Fd within the complex. 47 [30]. This corresponds to a vG³W30.1 eV for the ET step FXCFA. There is still much uncertainty 2.7. Intramolecular ET between [4Fe^4S] clusters of with regard to Em of FA and FB clusters in PSI. PSI. Can it be rate-limiting for Fd reduction? Values of about 3540 mV and 3590 mV have been found for FA and FB, respectively, by monitor- In the following, I will calculate the ET rates that ing EPR signals at low temperature. Higher values of one can expect for both steps FXCFA or FACFB about 3480 mV have been measured from room for di¡erent values of the change in Gibbs energy temperature experiments (see [10] for discussion). vG³ and of the reorganization energy V. There is By measuring decay of P700 at 820 nm, values of no available data on the Ems of PSI clusters and of 3440 mV and 3465 mV have been recently deter- Fd within the PSI/Fd complex. Therefore one must

BBABIO 45079 12-10-01 Cyaan Magenta Geel Zwart P. Se¨tif / Biochimica et Biophysica Acta 1507 (2001) 161^179 167

consider the possibility that Ems are modi¢ed due to Fd binding and this analysis will take such an e¡ect into account by assuming di¡erent values of vG³ for both steps: 30.1 and 0 eV for FXCFA, 0, +0.1 and +0.2 eV for FACFB. The reorganization energy V will be taken between 0.4 and 1.0 eV and I will use the optimal ET rates calculated in [34], i.e., 6.3U107 and 4U109 s31, for the two successive steps. In the framework of the semiclassical ET theory [35], the 2 electron transfer rate kET is given by: ket =(4Z / 31=2 2 2 h)(4ZVkT) MTABM exp[3(vG³+V) /4VkT] with TAB being the electronic coupling. The dependence of kET upon V given in Fig. 3 is calculated by neglect- ing the term (4ZVkT)31=2, which has a relatively small impact compared to the term (exp[-(vG³+V)2/ 4VkT]). kET decreases by almost one order of magnitude when V increases by 0.2 eV or when vG³ increases by 0.1 eV. A t1=2 of 4.5 Ws, which corresponds to the slowest ¢rst-order phase of Fd reduction in C. reinhardtii, is obtained, e.g., for (vG³, V)= (0.0 eV, 0.6 eV) and (0.1 eV, 0.8 eV) for the steps FXCFA and FACFB, respectively. Slightly larger values would give a t1=2W15 Ws, which is the intermediate ¢rst-order phase found in Synechocystis 6803. From this crude analysis, it appears that one must seriously consider the possibility that intramolecular PSI ET is responsible for the microsecond components of Fd reduction. If this is the case, one must also assume di¡erent conformational states in order to explain heterogeneity in intramolecular PSI ET. Fig. 3. Dependence of intramolecular PSI ET rates upon the reorganization energy V. Halftimes for the two steps F CF Two cases for this heterogeneity can be distinguished: X A and FACFB were calculated for di¡erent values of vG³. The it can be either intrinsic to PSI (whether Fd is bound vG³ values (given in eV) are indicated in the right part of the or not) (case 1), or associated to Fd binding (case 2). curves (0.0 and 30.1 eV for FXCFA ; 0.2, 0.1 and 0.0 eV for Case 1. A heterogeneity intrinsic to PSI might e.g., FACFB) (see text for details). arise from a di¡erent distribution of the mixed valence pair on one or both of the reduced clusters presence of Fd. In this case, heterogeneity in ET between which ET occurs [36]. This could result in means that the PSI/Fd complexes are signi¢cantly di¡erent electronic couplings or in di¡erent nuclear di¡erent in electrostatic interactions in order to factors (e.g., via di¡erent vG³). Redox potentials of give rise to various vG³ for any of the two ET steps iron^sulfur clusters might also be a¡ected by other FXCFA or FACFB. At the moment, there is no modi¢cations in the local environment. In the para- experimental data against this model. graph below, I will summarize experimental evidence obtained from the study of mutants which makes 2.8. Available evidence from Fd and PSI mutants for such a case unlikely. the origin of multiple ¢rst-order components of Case 2. Solvent screening within the complex and Fd reduction (or) electrostatic e¡ects due to the acidic residues of Fd may increase vG³ of one or both steps FXCFA In a PsaC mutant from C. reinhardtii, where two or FACFB and make these ET steps slower in the basic residues located close to FA have been mutated

BBABIO 45079 12-10-01 Cyaan Magenta Geel Zwart 168 P. Se¨tif / Biochimica et Biophysica Acta 1507 (2001) 161^179

(K52S/R53A), FB is preferentially photoreduced at plitudes of the submicrosecond and the intermediate low temperature contrarily to WT [19]. However, 20 Ws phase, with values of the ratio AsubWs/A20 Ws the kinetics of Fd reduction are not signi¢cantly af- comprised between 0.8 and 3.5 for simple mutants fected in the mutant, suggesting that ET between FA (W1.0^1.25 for WT Fd, see Table 2). Residues and FB has no in£uence on the rate of Fd reduction. D20, D57 and D60 are peripheral and relatively far However, this conclusion must be taken with much from the [2Fe^2S] cluster of Fd. If FB reduction dur- caution: ¢rstly, this has not been ascertained in the ing forward ET would be intrinsically (in the absence case of the submicrosecond component, which was of Fd) a multiphasic process, one would not expect not time-resolved; secondly, the redox e¡ect on (FA, any signi¢cant change in the relative proportion of FB) brought by the mutation might be very weak as the phases when any of these three Fd residues is it has been probed only through EPR at 15 K modi¢ed. Therefore, it appears very likely that the (kTW1.3 meV). intermediate phase is not due to intramolecular PSI More convincing evidence is provided by another ET in the absence of Fd and is associated with Fd PsaC mutant from C. reinhardtii, in which three res- binding or reduction. idues close to the FB cluster have been modi¢ed As a summary, I think that the following state- (I12V/T15K/Q16R) [37]. In this mutant, Fd a¤nity ments can be reliably made. is increased 60-fold and moreover, the ¢rst-order kinetics are dominated ( s 90%) by a fast submicrosec- 1. The distal cluster FB is photoreduced in the sub- ond reduction (Table 1). By comparison, only one microsecond time range in PSI alone. This is in line third of Fd is reduced by WT PSI in the submicro- with most of the photovoltage measurements pub- second time range. One can thus infer that most of lished yet concerning FA reduction [22,24,25] and FB is reduced in the submicrosecond time range in implies submicrosecond reduction of FB from FA. the mutant. The question arises whether this can be extrapolated to WT PSI. In the mutant, photoreduc- 2. Heterogeneity of ET kinetics is an intrinsic property tion at low temperature (14 K) leads to spectra and of Fd reduction, and can be most probably ascribed 3 3 amounts of photoreduced FA and FB similar to WT. to di¡erent conformations of the PSI/Fd complex. 3 3 If one assumes that the FB /FA ratio is related to the However, the available data are not su¤cient to relative Ems of the clusters [10], this implies that the decide whether the microsecond kinetics observed redox potentials of FA and FB are identical in the in part of the complexes is due to some rate limita- WT and in the mutant. This supports an identical tion within some PSI complexes due to Fd binding intramolecular PSI ET in the WT and in the mutant, or to heterogeneity in ET from FB to Fd. and therefore a submicrosecond reduction of FB in WT PSI, in the absence of Fd. Photoreduction of mutants of Fd from Synecho- cystis 6803, in which aspartates 20, 57 and 60 have 2.9. PSI/Fd complexes: discrete complexes or been changed to several other residues, has been continuous distribution? studied by £ash-absorption spectroscopy [38]. A large variation has been observed in the relative am- One can ask the question whether there is a small

Table 2

Ratio between the amplitudes of the fast (t1=2 6 1 Ws) and intermediate (t1=2W20 Ws) phases of Fd reduction in Synechocystis 6803 Residue substituting aspartic acid position of Cysteine Alanine Arginine Lysine mutated aspartic acid 20 0.9 2.0 3.5 1.25 57 1.1 0.6 1.25 1.4 60 1.7 2.2 2.0 0.8 The ratio is 1^1.25 for WT Fd and was recalculated from [38] for di¡erent Fd mutants. The measurements were made at pH 8.0 and in the presence of 30 mM NaCl and 5 mM MgCl2.

BBABIO 45079 12-10-01 Cyaan Magenta Geel Zwart P. Se¨tif / Biochimica et Biophysica Acta 1507 (2001) 161^179 169 number of di¡erent PSI/Fd conformations corre- of the semiquinone form [46]. However, in some spe- sponding to separate energy minima or whether the cies, the redox potentials of the two waves might be kinetic complexity could be explained by assuming a rather close at pH 8 or above, values that can occur continuous distribution of conformations. The very in vivo under light, so that a physiological role for clearcut di¡erence in rates between the submicrosec- the oxidized-semiquinone couple cannot be excluded ond component and the microsecond component(s) [2]. Photoreduction of oxidized Fld to its fully re- indicates that the fastest component corresponds to a duced form under continuous illumination was distinct type of complex. The picture is less clear in shown to be helpful for in vitro measurement of the case of Synechocystis 6803 at pH 8.0 with two PSI activity [47]. microsecond components. These components were Four studies of the photoreduction kinetics of oxi- ¢rst identi¢ed using a global analysis owing to the dized Fld by WT PSI have been published so far, fact that the corresponding decay associated spectra using £ash-absorption spectroscopy [46,48^50]. In are signi¢cantly di¡erent [18]. This does not exclude the ¢rst study, which was made with Fld from the the possibility that a continuous distribution model cyanobacterium Anabaena sp. PCC 7119 and spinach can account for the microsecond reduction of Fd PSI, only photoreduction of oxidized Fld was ob- inasmuch as the geometrical distribution of Fd is served [48]. A two-step mechanism, involving com- su¤cient to give rise to various spectral character- plex formation followed by intracomplex ET, was istics of Fd reduction. invoked to interpret the concentration dependence of the reduction rate. The second-order rate of complex formation was calculated to be 3.6U107 3. Electron transfer from PSI to £avodoxin M31 s31. The intracomplex ET rate, corresponding to the asymptotic value of Fld reduction at saturat- Under conditions of iron deprivation, £avodoxin ing Fld concentrations, was found at 270 s31. Both (Fld) can substitute for ferredoxin (Fd) in most cya- parameters were measured at pH 7.0 and 10 mM nobacteria and some algae [2,3]. In these organisms, NaCl. Reduction of Fld was found to depend both Fld is often considered to be able to replace Fd in all on NaCl and MgCl2 concentrations. For both salts, of its functions [39], but this was found not to be the a biphasic dependence of the rate of Fld reduction case in several cyanobacteria [40,41]. Salt stress has was observed (increase at low concentrations fol- been also reported to induce Fld accumulation in lowed by decrease at high concentrations). The in- Synechocystis 6803 [42], which would be particularly crease at low salt concentrations was ascribed to a involved in cyclic ET [43]. Flavodoxin from photo- rearrangement occurring within an initially formed synthetic organisms belong to the long-chain type collision complex, and to inhibition of this rearrange- [2,39]. They contain FMN as a redox active pros- ment at low ionic strength due to too strong electro- thetic group and can undergo two successive single static forces. The decrease at high salt concentrations electron reductions. Oxidized Fld is ¢rst reduced to a was ascribed to a weaker electrostatic steering lead- protonated semiquinone form (FMNHc) and then ing to a smaller probability of formation of produc- reduced to a hydroquinone form (FMNH3). For £a- tive complexes. Similar kinetics of photoreduction of vodoxins found in photoautotrophic organisms, the oxidized Fld were reported in later studies. Two of corresponding redox potentials are in the 3180/3240 these involved both partners from the same cyano- mV region for the FMN/FMNHc couple (at pH 7) bacterium, either Synechococcus sp. PCC 7002 [46] or and in the 3370/3470 mV region for the FMNHc/ Synechocystis 6803 [49]. A last study was performed FMNH3 couple [44]. Reduction of the semiquinone with PSI from C. reinhardtii and Fld from Synecho- of Fld occurs therefore at about the same redox po- cystis 6803 [50]. It was proposed in [46] that the tential as reduction of Fd. This might imply that FMNHc formation, which is relatively slow com- only the second wave of Fld reduction is involved pared to semiquinone reduction (see below), might in shuttling electrons, in line with the EPR detection be rate-limited by protonation of FMN. of the semiquinone form in cyanobacterial cells in The second wave of Fld reduction by PSI (FMNHc darkness [45] and with the relatively high stability to FMNH3) was observed in [46,49,50], under con-

BBABIO 45079 12-10-01 Cyaan Magenta Geel Zwart 170 P. Se¨tif / Biochimica et Biophysica Acta 1507 (2001) 161^179 ditions where most of Fld was reduced to the semi- of PsaA/PsaB in Fd/Fld binding, but there is no quinone form before £ash excitation. In the two ho- available data on this topic up to now. mologous cyanobacterial systems [46,49], the initial fast phase, with a t1=2 = 10/13 Ws independent on the 4.1. Covalent complexes concentration of Fld, was followed by a slower phase 0 8 due to a second-order process (kon = 1.7/2.2U10 A chemical cross-linking approach was success- M31 s31). The size of the 10/13 Ws ¢rst-order phase fully applied in the characterization of the ferredox- was found to increase with the amount of Fld but to in-binding site of PSI, pointing in all cases the par- saturate at a value representing 60^73% of the total ticipation of the PsaD subunit [20,52^56]. Cross- signal. Several explanations were proposed for this linking of Fd with other PSI subunits, such as e¡ect [46]. (1) Some partial degradation of PSI. PsaC [20], PsaE and PsaH [56], has been also evi- This appears highly unlikely as PSI from Synecho- denced with eukaryotic PSI. Subunits PsaC, PsaD cystis 6803 was generally found to completely bind and PsaF were found to cross-link to Fld [49,57]. Fd, as seen by the kinetics of Fd reduction. (2) Some The PSI/Fd complex from Synechocystis 6803 was incomplete reduction of the semiquinone Fld from found to contain Fd and PSI at a molar ratio close 3 (FA,FB) , as discussed above in the case of Fd to 1 [52]. Fd can be entirely photoreduced within the c 3 (Em = 3415 mV for the FMNH /FMNH couple in complex but only 60% of it was reduced with sub- Fld from Synechococcus 7002 (U. Mu«hlenho¡, W. microsecond and microsecond kinetics [58]. The PSI/ Nitschke, personal communication)). (3) The pres- Fld complex from Synechococcus 7002 was found to ence of a PSI/Fld complex not competent for ET be fully inhibited with respect to ET to soluble Fd, (27^40% of PSI), in slow equilibrium with the com- Fld, or FNR [59]. The kinetics of Fld photoreduction plex competent for fast ET. At present, there is no were studied in the covalent complex and involved available experimental evidence for favoring any of 40^50% of the complexes. Oxidized £avodoxin was 31 the two last possibilities. A dissociation constant (Kd) reduced with a rate of 145 s . The photoreduction of 2/2.6 WM was calculated when assuming a simple of semiquinone Fld exhibited three di¡erent compo- binding equilibrium (model 2) [46,49]. As discussed nents (t1=2W9 Ws, 70 Ws and 1 ms, [59]), which prob- with Fd, the 10^13 Ws component could correspond ably originate from some biochemical heterogene- c either to ET from FB to FMNH or to a rate-limiting ity. step within PSI when Fld is bound. It was also ob- The cyanobacterial PSI/Fd and PSI/Fld covalent served that the a¤nity of the semiquinone form of complexes were studied by electron microscopy com- Fld for PSI can be very poor in heterologous sys- bined with single particle image analysis. A similar tems, with no fast phase observed with Fld from docking site was found for Fd and Fld, which both Synechococcus 7002 and PSI from S. elongatus [49], bind to the stromal side of PSI, in close contact to a and with Fld from Synechocystis 6803 and PSI from ridge formed by the three subunits PsaC, PsaD and C. reinhardtii [50]. This is opposite to Fd binding, PsaE [58,59] (Fig. 4, lower position of Fd). These which was not found to be much dependent on ho- observations ¢t very well with a prediction for Fd mologous systems (Table 1; [17]). docking that was previously made from the PSI crys- tal structure [60]. In a recent report, the location of Fd was determined in spinach by Fourier di¡erence 4. The ferredoxin and £avodoxin binding sites of PSI analysis after cross-linking Fd to two-dimensional PSI crystals, which can be formed at the edges of It is now clearly established that the stromal ridge grana stacks [61]. In this study, Fd is found on top of PSI comprises the three subunits PsaC, PsaD and of the stromal ridge and appears to interact princi- PsaE [5,51]. I will discuss in this section the respec- pally with the PsaC and PsaE subunits (Fig. 4, upper tive roles of these three subunits inasmuch as reduc- position of Fd). A similar mode of Fd binding has tion of Fd and Fld and more generally ET properties been also inferred from the measurement of electric are concerned. This does not exclude the direct par- potentials by Kelvin force probe microscopy [62]. It ticipation of other subunits, e.g., some stromal loops was speculated in [61] that the mobility of Fd could

BBABIO 45079 12-10-01 Cyaan Magenta Geel Zwart P. Se¨tif / Biochimica et Biophysica Acta 1507 (2001) 161^179 171

Fig. 4. Scheme of the PSI stromal side. The scheme uses the X-ray model of PSI from S. elongatus [5] as a framework to incorporate the NMR structure of PsaE from Synechococcus 7002 [87] and the backbone structure of ferredoxin from Sulfolobus as a PsaC model [83]. Due to the present uncertainties concerning the PsaD subunit, only the general contour of this subunit is shown. Some essential residues of PsaC and PsaE are colored and numbered. The approximate positions of Fd within the PSI/Fd complexes are shown from the data obtained in Synechocystis 6803 (lower position, [58]) and spinach (upper position, [61]) (see text for further details). be hindered in cyanobacteria as a phycobilisome can al characterization of the spinach complex, apart reside on the stromal surface of PSI [63] whereas Fd from a partial inhibition of NADP photoreduction is freely accessible to the stroma in plants. This [61]. The spinach Fd location appears also hardly would have resulted in two di¡erent modes of Fd compatible with the large e¡ect on Kd brought by binding in the two di¡erent types of organism. In PsaC mutations in the vicinity of FB in the green the spinach complex, the minimal center-to-center alga C. reinhardtii ([37], Fig. 4, see below). Therefore distance between FB and the [2Fe^2S] cluster of Fd more stringent functional tests are required before was estimated at 11 Aî , which would be compatible one can ascertain that the conformation observed with fast reduction of Fd. However, this distance with spinach allows fast photoreduction of bound appears to be underestimated as the structural model Fd. One interesting alternative would be that a pre- shown in [61] does not take into account the recent liminary complex is trapped in spinach during cross- crystallographic PSI structure with the PsaC central linking, whereas this does not occur in Synechocystis loop covering the clusters [5]. There is little function- 6803.

BBABIO 45079 12-10-01 Cyaan Magenta Geel Zwart 172 P. Se¨tif / Biochimica et Biophysica Acta 1507 (2001) 161^179

4.2. Studies of PsaD- or PsaE-deleted mutants in the above model receives considerable support from site-directed mutations of PsaE from which it The PsaC subunit binds the terminal acceptors appears that a single arginine residue is entirely re- (FA,FB) of PSI and cannot be deleted without loss sponsible for the kinetic stabilization of the complex of PSI activity. This is not the case for the PsaD and (see below). After deletion of PsaE in Synechocystis PsaE subunits which presumably do not carry any 6803, photoreduction of oxidized Fld is little a¡ected cofactor. I will ¢rst discuss the e¡ects of PsaD or (2-fold increase compared to WT) whereas photore- PsaE deletion on the individual steps of Fd and duction of the semireduced Fld is modi¢ed in parallel Fld reduction (see also Table 1). Reduction of Fd to Fd reduction [49]: the second-order rate constant by PSI from Synechocystis 6803 is severely impaired is decreased by only 30% whereas no detectable fast in the absence of either PsaD or PsaE [12,13,64]. ¢rst-order reduction is observed. Both PsaD-minus However, in both cases, it was later recognized that and PsaE-minus PSI can be reconstituted with the Fd can be photoreduced with both submicrosecond respective missing subunits and the WT kinetic be- and microsecond kinetics at high Fd concentrations havior was essentially recovered both for Fd and Fld [65]. These fast kinetics correspond to ¢rst-order photoreductions [13,49,64,65]. By titrating the £uo- components, therefore indicating that Fd can bind rescence quenching of the isolated PsaD subunit by to PSI in the absence of PsaD or PsaE, albeit with Fd, both from spinach, a Kd in the nanomolar range a much decreased a¤nity. This is compatible with was found for the PsaD/Fd complex [67]. This very the observation that PsaD is not involved in orient- high a¤nity is in complete discrepancy with the Kd ing PsaC on the reaction center core, as seen by EPR (W50 WM) than can be measured in the absence of 3 3 detection of FA and FB in partially oriented samples PsaE in the cyanobacterium Synechocystis 6803. A [66]. For PsaD-less and PsaE-less PSI, Kds of the nanomolar Kd is also smaller than the value obtained PSI/Fd complex are increased. 25- and 100-fold, re- with WT PSI and Fd from spinach (in the micro- spectively, whereas second-order rate constants of Fd molar range; P. Se¨tif, unpublished observation). reduction are decreased 10- and 2-fold, respectively Moreover, PsaC has been shown to be primarily in- ([65], Table 1). From these measurements, changes in volved in Fd binding to chloroplastic PSI (see dis- the dissociation rate constant koff brought by both cussion of site-directed mutants from C. reinhardtii deletions were calculated by using the relation: below). Therefore a PsaD/Fd complex, with an a¤n- koff = KdUkon. Such a derivation assumes that there ity in the nanomolar range, seems not related to the is no large e¡ect of (FA,FB) single reduction on the physiological PSI/Fd complex. 0 second-order kinetics of Fd reduction, i.e., kon = kon The e¡ects on electron transport of the deletion of (Fig. 2). This assumption, though unproven, can be the psaD or psaE genes can be probed by other meth- justi¢ed by considering the large number of charges ods than the direct kinetic measurements of Fd or borne by the stromal surface of PSI. This should Fld reduction. I will brie£y summarize such measure- make electrostatic steering of Fd relatively insensitive ments in the following. To my knowledge, the psaD to a change of one unit charge, distributed on (FA, gene has been deleted only in the cyanobacterium FB) and hence not at the PSI surface. In this frame- Synechocystis 6803 [64,66,68,70]. This deletion leads work, e¡ects of PsaD and PsaE deletion lead to in- to a decrease in growth rates, but variable extents of creases in koff of about 2.5- and 50-fold, respectively this decrease were reported, from 50% to more than [65]. It was then proposed that PsaD is important for 90%, depending on the conditions ( þ glucose, light steering electrostatically Fd to its binding site (main intensity). With isolated membranes from the mu- 0 e¡ect on kon) whereas PsaE controls the lifetime of tant, NADP photoreduction amounts to 0^10% the PSI/Fd complex (main e¡ect on koff ) [65]. Thus and 10^30% that of WT with Fd and Fld, respec- di¡erent kinetic e¡ects of PsaD and PsaE deletions tively, whereas Fld photoreduction is 20^50% that of might result in similar Kds.The role of PsaD in this WT [66,70]. It appears therefore that Fld reduction is model is still needed to be con¢rmed by studies of less sensitive to the absence of PsaD than Fd reduc- point mutations. However, the speci¢c role of PsaE tion. Due to the fact that PsaD is essential for the

BBABIO 45079 12-10-01 Cyaan Magenta Geel Zwart P. Se¨tif / Biochimica et Biophysica Acta 1507 (2001) 161^179 173

PsaC stability [71], degradation of PSI during isola- PSI under continuous illumination: whereas a 35% tion may be partly responsible for the decrease in decrease was reported for photoreduction of re- photoreduction rates. combinant Fld by PSI, both from Synechococcus Deletion of PsaE was performed in several cyano- 7002 [47], no e¡ect was observed in the case of Syn- bacterial strains [13,69,72,73] as well as in Arabidop- echocystis 6803 [49]. sis [74]. The Arabidopsis mutants exhibit a marked increase in light sensitivity and photoinhibition, 4.3. Is a high a¤nity complex between Fd (Fld) and which may be ascribed to the signi¢cant loss of the PSI of any physiological importance? PsaC and PsaD subunits due to PSI destabilization [74]. In cyanobacteria, no e¡ect of psaE deletion on The PsaD subunit appears to be essential for the the growth rate was observed under conditions of stability of the PSI complex (see, e.g., [78^80]) where- photoautotrophic growth [69,72,73]. However, a as PsaE may have multiple roles, as described above large decrease in growth rate was observed in Syn- (stabilization of PSI, cyclic ET, involvement in a ter- echococcus 7002 under conditions of photoheterotro- nary complex with FNR). However, from the avail- phic growth and this e¡ect was attributed to impair- able experimental data, it is still not yet clear whether ment of cyclic ET [72,73]. In the same strain, the the role of both subunits in increasing the a¤nity of absence of PsaE was found to result in a de¢cient the PSI/Fd (or Fld) complexes, and hence in promot- bicarbonate uptake, which was ascribed to an im- ing fast reduction of Fd (Fld) is of any physiological paired cyclic ET [75]. In a recent photoacoustic importance. In this context, it may be useful to esti- study, the essential role of PsaE in cyclic ET was mate the rate that is needed for an e¤cient reduction questioned [76]. This was based on the fact that the of soluble acceptors. In photosynthetic reaction cen- energy storage is the same in WT and PsaE-deleted ters, the ET step which stabilizes a charge-separated Synechococcus 7002 when grown under photoauto- state is 2^3 orders of magnitude faster than the trophic conditions. However, one can ask to what wasteful recombination reaction. After full charge extent energy storage is relevant for probing cyclic separation within PSI, P700 can recombine with 3 ET as di¡erent electron pathways operating þ PsaE (FA,FB) with t1=2 of 30^100 ms. A rate faster by could lead to a similar energy storage. With isolated three orders of magnitude gives a t1=2 of 30^100 Ws. 3 membranes from the psaE deleted Synechocystis 6803 Stabilization of (P700 -(FA,FB) ) can be performed mutant, NADP photoreduction amounts to 5% and either via reduction of P700 by the soluble donors 35% that of WT with Fd and Fld, respectively, plastocyanin or cytochrome c6 or via reoxidation of 3 whereas Fld photoreduction is 50% that of WT (FA,FB) by the soluble acceptors Fd or Fld. Re- [70]. As is the case with PsaD, Fld reduction appears duction of P700 by soluble donors appears to be therefore to be less sensitive to the absence of PsaE relatively slow in some cyanobacteria due to the poor than Fd reduction. In a more recent study performed a¤nity of soluble donors for PSI [81]. Moreover, with a similar psaE minus mutant from Synechocystis part of P700 reduction always involves some slower 6803, NADP photoreduction with Fd was found to phase with t1=2 of hundreds of microseconds or lon- represent 50^100% that of the WT value [77]. From ger. Hence, Fd (Fld) reduction should compete e¤- simulations of PSI-dependent electron transport, it ciently with the recombination reaction between 3 was also found that the changes in the Fd reduction P700 and (FA,FB) , and this would imply a t1=2 kinetics due to the absence of PsaE, are not expected of 30^100 Ws. Furthermore a high e¤ciency of Fd to in£uence signi¢cantly NADP photoreduction (Fld) may be required for avoiding reduction of oxy- 3 [77]. However, the FNR expression level appeared gen from (FA,FB) which is potentially harmful for to increase in the mutant and the kinetic data sug- PSI. A t1=2 of 30^100 Ws corresponds to the slowest gested that the a¤nity of Fd for FNR is modulated ¢rst-order components observed in PSI/Fd com- by the presence of PsaE, in line with the existence of plexes and is slightly slower than what is observed a transient ternary complex PSI/Fd/FNR [77]. Con- for ¢rst-order reduction of semireduced Fld. In this troversial results were obtained concerning the e¡ect context, a very fast submicrosecond photoreduction of PsaE deletion on Fld photoreduction by isolated of Fd appears to be useless but it seems to have been

BBABIO 45079 12-10-01 Cyaan Magenta Geel Zwart 174 P. Se¨tif / Biochimica et Biophysica Acta 1507 (2001) 161^179

conserved in any type of organism tested yet. One vicinity of FB, a 60-fold increase in Fd a¤nity was can also estimate the concentration of soluble accep- observed, due probably to the introduction of two tor that is needed for getting a t1=2 of 100 Wsbya positive charges at the Fd binding site [37]. The 0 8 second-order process: assuming konW2U10 ¢rst-order kinetics of Fd reduction were also modi- M31 s31, one has [Fd] (or [Fld])W35 WM. The pre- ¢ed with a dominant submicrosecond phase. Two 0 ceding kon value is based on measurements made in other mutants were generated, with an acidic residue vitro with aqueous media. In vivo, viscosity is cer- being substituted for a neutral or a basic residue in tainly much higher. This will lead to a large decrease the vicinity of either FA or FB [37]. When E46 (close 0 in kon and photoreduction of Fd (Fld) through a to FA) was changed for Q or K, no signi¢cant second-order process might be ine¤cient. One can change in reduction of Fd was found [37]. When therefore speculate that in vivo complex formation D9 (close to FB) was changed for N or K, a small is useful, though not critical, for promoting e¤cient increase (1.3^2-fold) of Fd a¤nity was observed with 0 reduction of the soluble acceptors and avoiding re- no change in the association rate kon. 3 duction of oxygen by (FA,FB) . From in vivo degenerate oligonucleotide-directed mutagenesis of the internal loop of PsaC containing 4.4. Site-directed mutagenesis K35, it was found that this residue plays a key role in the electrostatic interaction between PSI and Fd [20]. Except when indicated, all mutated residues which Mutations K35T, K35D and K35E drastically a¡ect are explicitly mentioned below are highly conserved. Fd binding, which could not be observed (increase of Kd by at least 35-fold). More surprisingly, these sin- 4.4.1. PsaC gle mutations lead to a strong decrease in the asso- 0 E¡ects of PsaC site-directed mutations on Fd (Fld) ciation rate kon (5^82-fold). The mutants K35D and photoreduction have been studied with PSI from K35E of C. reinhardtii were also studied with Fld C. reinhardtii. Two categories of mutants were from Synechocystis 6803 [50]. Compared to WT studied: the ¢rst one, for which only Fd reduction PSI, only a slight decrease was observed for photo- was studied [19,37], was concerned with residues sur- reduction of oxidized Fld. The second-order reduc- rounding the cysteine ligands of the two clusters FA tion of the Fld semiquinone was much more a¡ected and FB ([82] and references therein); the second one, with a decrease by a factor of 10^19 compared to for which both Fd and Fld reductions were studied WT. This is in line with the observations made [20,50], was concerned with a lysine residue (K35) with Fd and indicates that K35 plays a key role in which belongs to an internal loop constituting an the electrostatic steering of both Fd and Fld. extension compared to most 2U[4Fe^4S] bacterial The 2U[4Fe^4S] ferredoxin from Sulfolobus con- ferredoxins. tains, similarly to PsaC, a 10-residues internal loop In PsaC of C. reinhardtii, cysteines (21, 48, 51, 54) compared to most bacterial ferredoxins. A model of and (11, 14, 17, 58) coordinate FA and FB, respec- the PsaC subunit backbone was build [37] from the tively. Di¡erent mutations of the two conserved basic structure of the Sulfolobus ferredoxin [83], the posi- residues K52 and R53, in the vicinity of FA, led to tion of the two short K-helices adjacent to the clus- destabilization of PSI but PSI from the two mutants, ters in the PSI structure [4] and with FB taken as the (K52S/R53A) and (V49I/K52T/R53Q), could be pu- distal cluster. A similar model is presented in Fig. 4 ri¢ed. (K52S/R53A) was the only PsaC mutant that in a another direction parallel to the membrane and was studied with Fd from Synechocystis 6803 and no facing the stromal ridge of PSI. In this representa- modi¢cation of the kinetics of Fd reduction was ob- tion, Fd presumably binds to PSI from the side of served [19]. The mutant (V49I/K52T/R53Q) was the ridge as deduced from electron microscopy of the studied with Fd from C. reinhardtii and a small de- covalent complex from Synechocystis 6803 ([58]; low- crease in a¤nity (3-fold) was observed, with no sig- er position in Fig. 4). The upper Fd position shown ni¢cant change in the rates of ¢rst-order or second- in Fig. 4 corresponds to the approx. position ob- order components compared to WT PSI. In the triple served in the spinach covalent complex [61]. The po- mutant (I12V/T15K/Q16R), with substitutions in the sitions of K35, T15 and Q16 in the backbone were

BBABIO 45079 12-10-01 Cyaan Magenta Geel Zwart P. Se¨tif / Biochimica et Biophysica Acta 1507 (2001) 161^179 175 determined by reference to the positions of the cys- mutant [13]. Among all mutants tested so far which teine ligands and are colored in Fig. 4. Residue K35 are not a¡ected in PSI assembly, the kinetics of Fd on the one hand, T15 and Q16 (residues mutated in photoreduction by isolated PSI were strongly dis- the high-a¤nity mutant I12V/T15K/Q16R) on the turbed only for mutants of R39 [86]. Four di¡erent other hand, appear to be located within the Fd bind- mutants, R39K, R39H, R39Q and R39D, were gen- ing domain with Fd in its lowest position. erated. Mutants R39K, R39Q and R39D exhibit 7-, 250- and s 450-fold decreases in a¤nity for Fd, re- 4.4.2. PsaD and PsaE spectively. A pH titration was performed for R39H, In the PSI/Fd covalent complex prepared from which shows that protonation of histidine is essential Synechocystis 6803, K106 from PsaD was found to for a good Fd a¤nity (by comparison to WT, 5- and be cross-linked to E93 of Fd [52]. This lysine and 140-fold decreases in a¤nity at pHs 5.8 and 8.0, some residues surrounding it were mutated in Syn- respectively). In the R39Q mutant, the association 0 echocystis 6803 [64,66]. K106 was substituted for six rate kon was decreased 3-fold compared to WT, di¡erent residues (R, D, E, N, S, G) in [66]. Fld whereas an 80-fold increase is calculated for the dis- photoreduction and Fld-mediated NADP photore- sociation rate. This behavior is very similar to that of duction were not modi¢ed in the mutants. Fd-medi- PsaE-minus PSI. It was then proposed [86] that a ated NADP photoreduction was decreased by 10^ major role of PsaE is to provide a positive charge 54%, with the largest decrease (W50%) occurring at position 39 and that this positive charge controls with charge reversal (mutants K106D and K106E). the lifetime of the complex (rate koff ) with Fd, in The kinetics of Fd photoreduction were also studied support of the model hypothesized from the deleted by £ash-absorption spectroscopy for mutants mutant [65]. The model shown in Fig. 4 incorporates K106R, K106A and K106C [64]. By comparison to the average structure of isolated PsaE from Synecho- WT, the pattern of ¢rst-order kinetics, i.e., the rela- coccus 7002 [87], in a position similar to that found tive proportions of the three phases, was little af- in the X-ray structure [5]. The side chain of R39 fected and a 1.5^4.5-fold decrease in a¤nity was re- appears to be exposed to the stroma and in a favor- ported. Therefore both studies point to a signi¢cant, able position to interact with Fd, when Fd is placed though not essential, role of K106. Residues H97 in the approximate position determined from the and R111 were also mutated and 2^8-fold decreases Synechocystis 6803 covalent complex (lower Fd posi- in a¤nity were observed [64]. Some changes in the tion in Fig. 4). kinetic pattern were also reported (e.g., almost no intermediate phase in H97E, a major slow phase in 4.4.3. Ferredoxin and £avodoxin R111C), indicating that this region is important for The fast kinetics of Fd photoreduction were modulating the relative proportions of the di¡erent probed by £ash-absorption spectroscopy only with PS/Fd complexes. The E93^95/Q93^95 mutant of Fd a few mutants, which were all generated from Syn- from Synechococcus elongatus (corresponding to 92^ echocystis 6803 [38,41]. No change was observed for 94 in Synechocystis 6803) can still cross-link to the C85V and E88A mutants ([41]; C85, which is not PsaD subunit of PSI [84]. This indicates the existence highly conserved, is putatively involved in a disul¢de of another electrostatic contact with PsaD, which bridge in Fd from Synechocystis 6803 [88]; an acidic may involve a region away from K106 in the primary residue is not fully conserved at position 88). A sequence. Other PsaD mutants were generated which 2-fold decrease in a¤nity was found for the E93A were mostly a¡ected in the assembly of PsaD into the mutant [41] (E93 can be cross-linked to K106 of PSI complex but no other residue directly responsible PsaD). This observation is consistent with a similar for Fd binding was identi¢ed [85]. e¡ect observed with the K106A mutant of PsaD [64]. Site-directed mutants of the PsaE subunit were A 10-fold decrease in a¤nity was observed with a Fd obtained only in Synechocystis 6803 [13,86], most mutant with a C-terminal extension of seven resi- mutations concerning basic residues. Some of the dues, although the ¢rst-order kinetics remained iden- mutants were impaired for PsaE integration into tical to those of the WT. This indicates that the PSI, with phenotypes identical to the psaE-deleted C-terminus extension has no e¡ect on the Fd posi-

BBABIO 45079 12-10-01 Cyaan Magenta Geel Zwart 176 P. Se¨tif / Biochimica et Biophysica Acta 1507 (2001) 161^179 tioning onto PSI [41]. As described above, mutations Fromme, H.T. Witt, W. Saenger, Photosystem I, an im- of D20, D57 and D60 lead to changes in the ¢rst- proved model of the stromal subunits PsaC, PsaD, and order kinetic pattern (Table 2) [38]. Therefore these PsaE, J. Biol. Chem. 274 (1999) 7351^7360. [6] R. Morales, M.H. Charon, G. Hudry-Clergeon, Y. Pe¨tillot, residues appear to be important for controlling the S. Norager, M. Medina, M. Frey, Re¢ned X-ray structures relative proportions of the di¡erent PS/Fd com- of the oxidized, at 1.3 Aî , and reduced, at 1.17 Aî , [2Fe^2S] plexes. Single mutations were found to give rise to ferredoxin from the cyanobacterium Anabaena PCC7119 a moderate decrease in a¤nity (1.25- to 6-fold) show redox-linked conformational changes, Biochemistry whereas a larger decrease (14- to 19-fold) was found 38 (1999) 15764^15773. [7] S. Im, G.H. Liu, C. Luchinat, A.G. Sykes, I. Bertini, The for the double mutants D57R/D60R and D57K/ solution structure of parsley [2Fe^2S] ferredoxin, Eur. D60K [38]. At the moment, almost nothing is known J. Biochem. 258 (1998) 465^477. about the precise geometry of the PSI/Fd and PSI/ [8] C.L. Drennan, K.A. Pattridge, C.H. Weber, A.L. Metzger, Fld complexes. The residues of PSI and Fd facing D.M. Hoover, M.L. Ludwig, Re¢ned structures of oxidized each other are not identi¢ed with the exception of £avodoxin from Anacystis nidulans, J. Mol. Biol. 294 (1999) K106 of PsaD and E93 in Fd (numbering from Syn- 711^724. [9] D.B. Kna¡, M. Hirasawa, Ferredoxin-dependent chloroplast echocystis 6803). No report on Fld mutants has ap- enzymes, Biochim. Biophys. Acta 1056 (1991) 93^125. peared as yet. The functional equivalence of Fd and [10] K. Brettel, Electron transfer and arrangement of the redox Fld has been investigated on a structural basis by cofactors in photosystem I, Biochim. Biophys. Acta 1318 aligning the electrostatic potentials of both proteins (1997) 322^373. [89]. Two di¡erent alignments with a very high de- [11] B. Ke, Primary electron acceptor of photosystem I, Biochim. Biophys. Acta 301 (1973) 1^33. gree of similarity in the electrostatic potential were [12] K. Sonoike, H. Hatanaka, S. Katoh, Small subunits of pho- proposed. tosystem I reaction center complexes from Synechococcus elongatus. II. The psaE gene product has a role to promote interaction between the terminal electron acceptor and fer- Acknowledgements redoxin, Biochim. Biophys. Acta 1141 (1993) 52^57. [13] F. Rousseau, P. Se¨tif, B. Lagoutte, Evidence for the involvement of PSI-E subunit in the reduction of ferredoxin by I am grateful to Patrick Jordan, Drs. Norbert photosystem I, EMBO J. 12 (1993) 1755^1765. Krauss, Jean-Marc Moulis and Ulrich Mu«hlenho¡ [14] A.A. Mamedova, M.D. Mamedov, K.N. Gourovskaya, I.R. for communicating unpublished data, and Drs. Vassiliev, J.H. Golbeck, A.Y. Semenov, Electrometrical Herve¨ Bottin and Bernard Lagoutte for discussions. study of electron transfer from the terminal FA/FB iron^sulfur clusters to external acceptors in photosystem I, FEBS Lett. 462 (1999) 421^424. [15] M. Hervas, J.A. Navarro, G. Tollin, A laser £ash spectros- References copy study of the kinetics of electron transfer from spinach photosystem I to spinach and algal ferredoxins, Photochem. [1] D.B. Kna¡, Ferredoxin and ferredoxin-dependent enzymes, Photobiol. 56 (1992) 319^324. in: D.R. Ort, C. Yocum (Eds.), Oxygenic Photosynthesis: [16] J.A. Navarro, M. Hervas, C.G. Genzor, G. Cheddar, M.F. The Light Reactions, Kluwer Academic Publishers, Dor- Fillat, M.A. De la Rosa, C. Gomez-Moreno, H. Cheng, B. drecht, 1996, pp. 333^361. Xia, Site-speci¢c mutagenesis demonstrates that the structur- [2] L.J. Rogers, Ferredoxins, £avodoxins and related proteins: al requirements for e¤cient electron transfer in Anabaena structure, function and evolution, in: P. Fay, C. Van Baalen ferredoxin and £avodoxin are highly dependent on the reac- (Eds.), The Cyanobacteria, Elsevier, Amsterdam, 1987, tion partner: kinetic studies with photosystem I, ferre- pp. 35^67. doxin:NADP reductase, and cytochrome c, Arch. Biochem. [3] N.A. Straus, Iron deprivation: physiology and gene regula- Biophys. 321 (1995) 229^238. tion, in: D.A Bryant (Ed.), The Molecular Biology of Cya- [17] P. Se¨tif, H. Bottin, Laser £ash absorption spectroscopy nobacteria, Kluwer Academic Publishers, Dordrecht, 1994, study of ferredoxin reduction by photosystem I in Synecho- pp. 731^750. cystis sp. PCC 6803: evidence for submicrosecond and mi- [4] W.D. Schubert, O. Klukas, N. Krauss, W. Saenger, P. crosecond kinetics, Biochemistry 33 (1994) 8495^8504. Fromme, H.T. Witt, Photosystem I of Synechococcus elon- [18] P. Se¨tif, H. Bottin, Laser £ash absorption spectroscopy gatus at 4 Aî resolution: comprehensive structure analysis, study of ferredoxin reduction by photosystem I: spectral J. Mol. Biol. 272 (1997) 741^769. and kinetic evidence for the existence of several photosystem [5] O. Klukas, W.D. Schubert, P. Jordan, N. Krauss, P. I-ferredoxin complexes, Biochemistry 34 (1995) 9059^9070.

BBABIO 45079 12-10-01 Cyaan Magenta Geel Zwart P. Se¨tif / Biochimica et Biophysica Acta 1507 (2001) 161^179 177

[19] N. Fischer, P. Se¨tif, J.-D. Rochaix, Targeted mutations in Garab (Ed.), Photosynthesis: Mechanisms and E¡ects, Vol. the psaC gene of Chlamydomonas reinhardtii: preferential I, Kluwer Academic Publishers, Dordrecht, 1998, pp. 663^

reduction of FB at low temperature is not accompanied by 666. altered electron £ow from photosystem I to ferredoxin, Bio- [32] R. Cammack, K.K. Rao, C.P. Bargeron, K.G. Hutson, P.W. chemistry 36 (1997) 93^102. Andrew, L.J. Rogers, Midpoint redox potentials of plant [20] N. Fischer, M. Hippler, P. Se¨tif, J.-P. Jacquot, J.-D. Ro- and algal ferredoxins, Biochem. J. 168 (1977) 205^209. chaix, The PsaC subunit of photosystem I provides an essen- [33] H. Bottin, B. Lagoutte, Ferredoxin and £avodoxin from the tial lysine residue for fast electron transfer to ferredoxin, cyanobacterium Synechocystis sp PCC 6803, Biochim. Bio- EMBO J. 17 (1998) 849^858. phys. Acta 1101 (1992) 48^56. [21] N. Krauss, W. Hinrichs, I. Witt, P. Fromme, W. Pritzkow, [34] O. Klukas, W.D. Schubert, P. Jordan, N. Krauss, P. Z. Dauter, C. Betzel, K.S. Wilson, H.T. Witt, W. Saenger, Fromme, H.T. Witt, W. Saenger, Localization of two phyl-

Three-dimensional structure of system I of photosynthesis at loquinones, QK and QKP, in an improved electron density 6Aî resolution, Nature 361 (1993) 326^331. map of photosystem I at 4-Aî resolution, J. Biol. Chem. [22] A. Diaz-Quintana, W. Leibl, H. Bottin, P. Se¨tif, Electron 274 (1999) 7361^7367. transfer in photosystem I reaction centers follows a linear [35] R.A. Marcus, N. Sutin, Electron transfers in chemistry and

pathway in which iron^sulfur cluster FB is the immediate biology, Biochim. Biophys. Acta 811 (1985) 265^322. electron donor to soluble ferredoxin, Biochemistry 37 [36] J.G. Huber, J. Gaillard, J.M. Moulis, NMR of Chromatium (1998) 3429^3439. vinosum ferredoxin: evidence for structural inequivalence [23] I.R. Vassiliev, Y.S. Jung, F. Yang, J.H. Golbeck, PsaC sub- and impeded electron transfer between the two [4Fe^4S]

unit of photosystem I is oriented with iron^sulfur cluster FB clusters, Biochemistry 34 (1995) 194^205. as the immediate electron donor to ferredoxin and £avodox- [37] N. Fischer, P. Se¨tif, J.-D. Rochaix, Site-directed mutagenesis

in, Biophys. J. 74 (1998) 2029^2035. of the PsaC subunit of photosystem I ^ FB is the cluster [24] W. Leibl, B. Toupance, J. Breton, Photoelectric character- interacting with soluble ferredoxin, J. Biol. Chem. 274 ization of forward electron transfer to iron^sulfur centers in (1999) 23333^23340. photosystem I, Biochemistry 34 (1995) 10237^10244. [38] I. Guillouard, B. Lagoutte, G. Moal, H. Bottin, Importance [25] M.D. Mamedov, R.M. Gadzhieva, K.N. Gourovskaya, L.A. of the region including aspartates 57 and 60 of ferredoxin on Drachev, A.Y. Semenov, Electrogenicity at the donor/accep- the electron transfer complex with photosystem I in the cy- tor sides of cyanobacterial photosystem I, J. Bioenerg. Bio- anobacterium Synechocystis sp PCC 6803, Biochem. Bio- membr. 28 (1996) 517^522. phys. Res. Comm. 271 (2000) 647^653. [26] K. Sigfridsson, O. Hansson, P. Brzezinski, Electrogenic light [39] S.G. Mayhew, G. Tollin, General properties of £avodoxins, reactions in photosystem I: resolution of electron-transfer in: F. Mu«ller (Ed.), Chemistry and Biochemistry of Flavoen- rates between the iron^sulfur centers, Proc. Natl. Acad. zymes, CRC Press, Boca Raton, FL, 1992, pp. 389^426. Sci. USA 92 (1995) 3458^3462. [40] J. Van der Plas, R. De Groot, M. Woortman, F. Cremers, [27] P. Kyritsis, J.G. Huber, I. Quinkal, J. Gaillard, J.M. Moulis, M. Borrias, G. Van Arkel, P. Weisbeek, Genes encoding Intramolecular electron transfer between [4Fe^4S] clusters ferredoxins from Anabaena sp. PCC 7937 and Synechococcus studied by proton magnetic resonance spectroscopy, Bio- sp. PCC 7942: structure and regulation, Photosynth. Res. 18 chemistry 36 (1997) 7839^7846. (1988) 179^204. [28] D. Bentrop, I. Bertini, C. Luchinat, W. Nitschke, U. Mu«h- [41] M. Poncelet, C. Cassier-Chauvat, X. Leschelle, H. Bottin, F.

lenho¡, Characterization of the unbound 2[Fe4S4]-ferredox- Chauvat, Targeted deletion and mutational analysis of the in-like photosystem I subunit PsaC from the cyanobacterium essential (2Fe^2S) plant-like ferredoxin in Synechocystis Synechococcus elongatus, Biochemistry 36 (1997) 13629^ PCC 6803 by plasmid shu¥ing, Mol. Microbiol. 28 (1998) 13637. 813^821. [29] M.L. Antonkine, D. Bentrop, I. Bertini, C. Luchinat, G.Z. [42] S. Fulda, M. Hagemann, Salt treatment induced accumula- Shen, D.A. Bryant, D. Stehlik, J.H. Golbeck, Paramagnetic tion of £avodoxin in the cyanobacterium Synechococcus sp. 1H NMR spectroscopy of the reduced, unbound photosys- PCC 6803, J. Plant Physiol. 146 (1995) 520^526. tem I subunit PsaC: sequence-speci¢c assignment of contact- [43] M. Hagemann, R. Jeanjean, S. Fulda, M. Havaux, F. Joset, shifted resonances and identi¢cation of mixed- and equal- N. Erdmann, Flavodoxin accumulation contributes to en- 3 3 valence Fe-Fe pairs in [4Fe^4S] centers FA and FB , hanced cyclic electron £ow around photosystem I in salt- J. Biol. Inorg. Chem. 5 (2000) 381^392. stressed cells of Synechocystis sp strain PCC 6803, Physiol. [30] V.P. Shinkarev, I.R. Vassiliev, J.H. Golbeck, A kinetic as- Plant 105 (1999) 670^678.

sessment of the sequence of electron transfer from FX to FA [44] G.A. Sykes, L.J. Rogers, Redox potentials of algal and cy- and further to FB in photosystem I: the value of the equi- anobacterial £avodoxins, Biochem. J. 44 (1984) 845^850. librium constant between FX and FA, Biophys. J. 78 (2000) [45] J.R. Norris, H.L. Crespi, J.J. Katz, Characterization of a 363^372. new photo-ESR signal associated with photosynthesis, Bio- [31] R. Jordan, U. Nessau, E. Schlodder, Charge recombination chem. Biophys. Res. Commun. 49 (1972) 139^146. between the reduced iron^sulphur clusters and P700,in:G. [46] U. Mu«hlenho¡, P. Se¨tif, Laser £ash absorption spectroscopy

BBABIO 45079 12-10-01 Cyaan Magenta Geel Zwart 178 P. Se¨tif / Biochimica et Biophysica Acta 1507 (2001) 161^179

study of £avodoxin reduction by photosystem I in Synecho- R.C. Ford, The location of the mobile electron carrier fer- coccus sp. PCC 7002, Biochemistry 35 (1996) 1367^1374. redoxin in vascular plant photosystem I, J. Biol. Chem. 275 [47] J.D. Zhao, R.G. Li, D.A. Bryant, Measurement of photo- (2000) 36250^36255. system I activity with photoreduction of recombinant £avo- [62] I. Lee, J.W. Lee, A. Stubna, E. Greenbaum, Measurement of doxin, Anal. Biochem. 264 (1998) 263^270. electrostatic potentials above oriented single photosynthetic [48] M. Medina, M.L. Peleato, E. Mendez, C. Gomez-Moreno, reaction centers, J. Phys. Chem. B 104 (2000) 2439^2443. Identi¢cation of speci¢c carboxyl groups on Anabaena PCC [63] J.J. van Thor, C.W. Mullineaux, H.C.P. Matthijs, K.J. Hel- 7119 £avodoxin which are involved in the interaction with lingwerf, Light harvesting and state transitions in cyanobac- ferredoxin-NADP reductase, Eur. J. Biochem. 203 (1992) teria, Bot. Acta 111 (1998) 430^443. 373^379. [64] J. Hanley, P. Se¨tif, H. Bottin, B. Lagoutte, Mutagenesis of [49] K. Meimberg, B. Lagoutte, H. Bottin, U. Mu«hlenho¡, The photosystem I in the region of the ferredoxin crosslinking PsaE subunit is required for complex formation between site: modi¢cations of positively charged amino acids, Bio- photosystem I and £avodoxin from the cyanobacterium Syn- chemistry 35 (1996) 8563^8571. echocystis sp. PCC 6803, Biochemistry 37 (1998) 9759^9767. [65] P. Barth, B. Lagoutte, P. Se¨tif, Ferredoxin reduction by [50] K. Meimberg, N. Fischer, J.D. Rochaix, U. Mu«hlenho¡, photosystem I from Synechocystis sp. PCC 6803: toward Lys35 of PsaC is required for the e¤cient photoreduction an understanding of the respective roles of subunits PsaD of £avodoxin by photosystem I from Chlamydomonas rein- and PsaE in ferredoxin binding, Biochemistry 37 (1998) hardtii, Eur. J. Biochem. 263 (1999) 137^144. 16233^16241. [51] J. Kruip, P.R. Chitnis, B. Lagoutte, M. Rogner, E.J. Boe- [66] V.P. Chitnis, Y.S. Jung, L. Albee, J.H. Golbeck, P.R. Chit- kema, Structural organization of the major subunits in cya- nis, Mutational analysis of photosystem I polypeptides. Role nobacterial photosystem I. Localization of subunits PsaC, - of PsaD and the lysyl 106 residue in the reductase activity of D, -E, -F, and -J, J. Biol. Chem. 272 (1997) 17061^17069. photosystem I, J. Biol. Chem. 271 (1996) 11772^11780. [52] C. Lelong, P. Se¨tif, B. Lagoutte, H. Bottin, Identi¢cation of [67] V. Pandini, A. Aliverti, G. Zanetti, Interaction of the soluble the amino acids involved in the functional interaction be- recombinant PsaD subunit of spinach photosystem I with tween photosystem I and ferredoxin from Synechocystis sp. ferredoxin I, Biochemistry 38 (1999) 10707^10713. PCC 6803 by chemical crosslinking, J. Biol. Chem. 269 [68] P.R. Chitnis, P.A. Reilly, N. Nelson, Insertional inactivation (1994) 10034^10039. of the gene encoding subunit II of photosystem I from the [53] G. Zanetti, G. Merati, Interaction between photosystem I cyanobacterium Synechocystis sp. PCC 6803, J. Biol. Chem. and ferredoxin. Identi¢cation by chemical crosslinking of 264 (1989) 18381^18385. the polypeptide which binds ferredoxin, Eur. J. Biochem. [69] P.R. Chitnis, P.A. Reilly, M.C. Miedel, N. Nelson, Structure 169 (1987) 143^146. and targeted mutagenesis of the gene encoding 8-kDa sub- [54] A.L. Zilber, R. Malkin, Ferredoxin cross-links to a 22 kD unit of photosystem I from the cyanobacterium Synechocys- subunit of photosystem I, Plant Physiol. 88 (1988) 810^814. tis sp. PCC 6803, J. Biol. Chem. 264 (1989) 18374^18380. [55] R.M. Wynn, J. Omaha, R. Malkin, Structural and function- [70] Q. Xu, Y.S. Jung, V.P. Chitnis, J.A. Guikema, J.H. Gol- al properties of the cyanobacterial photosystem I complex, beck, P.R. Chitnis, Mutational analysis of photosystem I Biochemistry 28 (1989) 5554^5560. polypeptides in Synechocystis PCC 6803. Subunit require- [56] B. Andersen, B. Koch, H.V. Scheller, Structural and func- ments for reduction of NADP, mediated by ferredoxin tional analysis of the reducing side of photosystem I, Phys- and £avodoxin, J. Biol. Chem. 269 (1994) 21512^21518. iol. Plant 84 (1992) 154^161. [71] N. Li, J. Zhao, P.V. Warren, J.T. Warden, D.A. Bryant, [57] U. Mu«hlenho¡, J. Zhao, D.A. Bryant, Interaction between J.H. Golbeck, PsaD is required for the stable binding of photosystem I and £avodoxin from the cyanobacterium Syn- PsaC to the photosystem I core protein of Synechococcus echococcus sp. PCC 7002 as revealed by chemical crosslink- sp. PCC 6301, Biochemistry 30 (1991) 7863^7872. ing, Eur. J. Biochem. 235 (1996) 324^331. [72] L. Yu, J. Zhao, U. Mu«hlenho¡, D.A. Bryant, J.H. Golbeck, [58] C. Lelong, E.J. Boekema, J. Kruip, H. Bottin, M. Ro«gner, P. PsaE is required for in vivo cyclic electron £ow around pho- Se¨tif, Characterization of a redox active crosslinked complex tosystem I in the cyanobacterium Synechococcus sp. PCC between cyanobacterial photosystem I and soluble ferredox- 7002, Plant Physiol. 103 (1993) 171^180. in, EMBO J. 15 (1996) 2160^2168. [73] J. Zhao, W.B. Synder, U. Mu«hlenho¡, E. Rhiel, P.V. War- [59] U. Mu«hlenho¡, J. Kruip, D.A. Bryant, M. Ro«gner, P. Se¨tif, ren, J.H. Golbeck, D.A. Bryant, Cloning and characteriza- E. Boekema, Characterization of a redox-active cross-linked tion of the psaE gene of the cyanobacterium Synechococcus complex between cyanobacterial photosystem I and its phys- sp PCC 7002: characterization of a psaE mutant and over- iological acceptor £avodoxin, EMBO J. 15 (1996) 488^497. production of the protein in Escherichia coli, Mol. Micro- [60] P. Fromme, W.D. Schubert, N. Krauss, Structure of photobiol. 9 (1993) 183^194. system I. Docking sites for plastocyanin and ferredoxin, and [74] C. Varotto, P. Pesaresi, J. Meurer, R. Oelmuller, S. Steiner- the coordination of P700, Biochim. Biophys. Acta 1187 Lange, F. Salamini, D. Leister, Disruption of the Arabidopsis (1994) 99^105. photosystem I gene psaE1 a¡ects photosynthesis and impairs [61] S.V. Ru¥e, A.O. Mustafa, A. Kitmitto, A. Holzenburg, growth, Plant J. 22 (2000) 115^124.

BBABIO 45079 12-10-01 Cyaan Magenta Geel Zwart P. Se¨tif / Biochimica et Biophysica Acta 1507 (2001) 161^179 179

[75] D. Su«ltemeyer, G.D. Price, D.A. Bryant, M.R. Badger, [84] B. Floss, G.L. Igloi, C. Cassier-Chauvat, U. Mu«hlenho¡, PsaE- and NdhF-mediated electron transport a¡ect bicar- Molecular characterization and overexpression of the petF bonate transport rather than carbon dioxide uptake in the gene from Synechococcus elongatus: evidence for a second cyanobacterium Synechococcus sp. PCC7002, Planta 201 site of electrostatic interaction between ferredoxin and the (1997) 36^42. PS I-D subunit, Photosynth.Res. 54 (1997) 63^71. [76] D. Charlebois, D. Mauzerall, Energy storage and optical [85] V.P. Chitnis, A. Ke, P.R. Chitnis, The PsaD subunit of cross-section of PS I in the cyanobacterium Synechococcus photosystem I. Mutations in the basic domain reduce the PCC 7002 and a psaE3 mutant, Photosynth. Res. 59 (1999) level of PsaD in the membranes, Plant Physiol. 115 (1997) 27^38. 1699^1705. [77] J.J. van Thor, T.H. Geerlings, H.C.P. Matthijs, K.J. Helling- [86] P. Barth, I. Guillouard, P. Se¨tif, B. Lagoutte, Essential role werf, Kinetic evidence for the PsaE-dependent transient ter- of a single arginine of photosystem I in stabilizing the elec- nary complex photosystem I/ferredoxin/ferredoxin:NADP tron transfer complex with ferredoxin, J. Biol. Chem. 275 reductase in a cyanobacterium, Biochemistry 38 (1999) (2000) 7030^7036. 12735^12746. [87] C.J. Falzone, Y.H. Kao, J. Zhao, D.A. Bryant, J.T.J. Le- [78] J.H. Golbeck, D.A. Bryant, Photosystem I, Curr. Top. Bio- comte, Three-dimensional solution structure of PsaE from energ. 16 (1991) 83^177. the cyanobacterium Synechococcus sp. strain PCC 7002, a [79] J.H. Golbeck, Photosystem I in cyanobacteria, in: D.A. Bry- photosystem I protein that shows structural homology with ant (Ed.), The Molecular Biology of Cyanobacteria, Kluwer SH3 domains, Biochemistry 33 (1994) 6052^6062. Academic Publishers, Dordrecht, 1994, pp. 319^360. [88] C. Lelong, P. Se¨tif, H. Bottin, F. Andre¨, J.-M. Neumann, 1H [80] P.R. Chitnis, Q. Xu, V.P. Chitnis, R. Nechushtai, Function and 15N NMR sequential assignment, secondary structure, and organization of photosystem I polypeptides, Photo- and tertiary fold of [2Fe^2S] ferredoxin from Synechocystis synth. Res. 44 (1995) 23^40. sp. PCC 6803, Biochemistry 34 (1995) 14462^14473. [81] M. Hervas, J.A. Navarro, A. Diaz, H. Bottin, M.A. De la [89] G.M. Ullmann, M. Hauswald, A. Jensen, E.W. Knapp, Rosa, Laser-£ash kinetic analysis of the fast electron transfer Structural alignment of ferredoxin and £avodoxin based on

from plastocyanin and cytochrome c6 to photosystem I. Ex- electrostatic potentials: implications for their interactions perimental evidence on the evolution of the reaction mecha- with photosystem I and ferredoxin-NADP reductase, Pro- nism, Biochemistry 34 (1995) 11321^11326. teins Struct. Funct. Genet. 38 (2000) 301^309. [82] J.H. Golbeck, A comparative analysis of the spin state dis- [90] R. Ku«mmerle, J. Gaillard, P. Kyritsis, J.-M. Moulis, Intra- tribution of in vitro and in vivo mutants of PsaC ^ A bio- molecular electron transfer in [4Fe^4S] proteins: estimates of chemical argument for the sequence of electron transfer in the reorganization energy and electronic coupling in Chro-

Photosystem I as FXCFACFBCferredoxin/£avodoxin, matium vinosum ferredoxin, J. Biol. Inorg. Chem. 6 (2001) Photosynth. Res. 61 (1999) 107^144. 446^451. [83] T. Fujii, Y. Hata, T. Wakagi, N. Tanaka, T. Oshima, Novel zinc-binding centre in thermoacidophilic archaeal ferredoxins, Nat. Struct. Biol. 3 (1996) 834^837.

BBABIO 45079 12-10-01 Cyaan Magenta Geel Zwart