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Biochimica et Biophysica Acta 1757 (2006) 339–345 http://www.elsevier.com/locate/bba

Review

Consequences of the structure of the b6 f complex for its charge transfer pathways ⁎ William A. Cramer , Huamin Zhang

Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA Received 17 January 2006; received in revised form 30 March 2006; accepted 24 April 2006 Available online 4 May 2006

Abstract

At least two features of the crystal structures of the cytochrome b6 f complex from the thermophilic cyanobacterium, Mastigocladus laminosus and a green alga, Chlamydomonas reinhardtii, have implications for the pathways and mechanism of charge (electron/proton) transfer in the complex: (i) The narrow 11×12 Å portal between the p-side of the quinone exchange cavity and p-side /quinol binding niche, through which all Q/QH2 must pass, is smaller in the b6 f than in the bc1 complex because of its partial occlusion by the phytyl chain of the one bound a molecule in the b6 f complex. Thus, the pathway for trans-membrane passage of the lipophilic quinone is even more labyrinthine in the b6 f than in the bc1 complex. (ii) A unique covalently bound , heme cn, in close proximity to the n-side b heme, is present in the b6 f complex. The b6 f structure implies that a Q cycle mechanism must be modified to include heme cn as an intermediate between heme bn and plastoquinone bound at a different site than in the bc1 complex. In addition, it is likely that the heme bn–cn couple participates in photosytem + I-linked cyclic electron transport that requires and the ferredoxin: NADP reductase. This pathway through the n-side of the b6 f complex could overlap with the n-side of the Q cycle pathway. Thus, either regulation is required at the level of the state of the that would allow them to be shared by the two pathways, and/or the two different pathways are segregated in the membrane. © 2006 Elsevier B.V. All rights reserved.

Keywords: Carbon fixation; Cytochrome b6 f, bc1 complexes; Cyclic electron transport; Heme cn; Q cycle

1. Introduction Mastigocladus laminosus (pdb id: stigmatellin and DBMIB complexes, 1VF5 and 2D2B, respectively), and the green alga Determination of the crystal structure of the cytochrome b6 f Chlamydomonas reinhardtii (pdb id: stigmatellin complex, complex at a resolution of 3.0 [1]–3.1 Å [2] completed the 1Q90), the structures of the other electron transport complexes description of the protein architecture responsible for electron that define the photosynthetic , the PSII transport and proton translocation in oxygenic . [3] and PSI [4] reaction centers, are derived from thermophilic With the exception of the b6 f complex, for which structures cyanobacteria. The high degree of similarity of the 3.1 Å and have been obtained from both the thermophilic cyanobacterium, 3.0 Å structures from M. laminosus and C. reinhardtii, the similarity of mass spectra of the complex from M. laminosus and spinach [5], and a large array of spectrophotometric and Abbreviations: BIC, butyl isocyanide; DBMIB, 2,5-dibromo-3-methyl-6- sequence data (not shown), indicates that the b6 f structure from isopropyl-p-benzoquinone; Δμ˜ +, trans-membrane proton electrochemical po- H the thermophilic cyanobacterium is representative of those in tential gradient; DOPC, dioleoyl-phosphatidylcholine; Em, midpoint oxidation- ; EPR, electron paramagnetic resonance; FNR, ferredoxin: green algae and probably higher plants as well. NADP+ reductase; ISP, - protein; NQNO, 2-n-nonyl-4-hydroxyquino- line N-oxide; p-and n-, electrochemically positive and negative sides of the 2. Structure of the complex membrane; PQ, lastoquinone; PS, ; TDS, tridecyl-stigmatellin; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TMH, trans-membrane helix The structure of the dimeric b6 f complex from M. laminosus ⁎ Corresponding author. Tel.: +1 765 494 4956; fax: +1 765 496 1189. contains 8 natural prosthetic groups per monomer (6 redox E-mail address: [email protected] (W.A. Cramer). groups and 2 pigment molecules) and consists of eight

0005-2728/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2006.04.020 340 W.A. Cramer, H. Zhang / Biochimica et Biophysica Acta 1757 (2006) 339–345 polypeptide subunits, which can be divided into two groups of four: (a) Four “large” (MW=17.5–32 kDa) core polypeptide subunits bind the redox prosthetic groups [four hemes, one [2Fe-2S] cluster, and a plastoquinone in M. laminosus], and participate in electron and proton transfer reactions. These subunits are f and b6 , the [2Fe-2S] Rieske iron- sulfur protein (ISP), and subunit IV, which provides the binding environment for the two additional prosthetic groups in each monomer, a chlorophyll a and β-carotene. (b) Four “small” (MW=3–4 kDa) subunits designated PetG, L, M, and N, each of which spans the complex and the membrane once, can be considered to be “hydrophobic sticks” forming a hydrophobic “picket fence”, with one-two C-terminal positively charged residues on the n-or stromal side of each of these subunits. The protein moiety of the eight subunit dimeric complex in M. laminosus has a molecular weight of 217 kDa [5]. Structures of protein components of the complex were previously obtained to a resolution of 1.9 Å for the C-terminal 250 residue extrinsic Fig. 1. Structure of the showing the eight-subunit soluble domain of cytochrome f [6], and of 1.83 Å for the N- dimeric organization. The color code is as follows: cytochrome b6( green); subunit IV (blue); cytochrome f (pink); ISP (yellow); PetG, -L, -M and -N terminal 139 residue soluble domain of the ISP [7,8]. (orange). The iron-sulfur cluster is shown as orange and purple spheres. Other

Cytochrome b6 and subunit IV, which define the core of the prosthetic groups are shown as sticks: heme f (pink); heme b (purple); heme cn complex (TM helices A-D and E-G, respectively), are highly (pink); PQ (blue); chlorophyll (green); β-carotene (orange); TDS (orange). homologous in their distribution of hydrophobic amino acids to the N-and C-terminal halves of in the bc1 complex [9]. cavity, first observed in the mitochondrial cytochrome bc1 complex [12], exchanges quinone-quinol with the bulk phase 2.1. An additional subunit in the b6 f complex from higher plant of the membrane. The lipophilic nature of the cavity ; the ferredoxin:NADP+ reductase was defined by the observation that two of the ten lipid molecules per monomer added to the complex for crystalli- The same eight subunits found in the b6 f complex from M. zation [17] reside in the cavity [1], and also presumably laminosus and C. reinhardtii are also found, with very similar contribute to its stability. The identification of the cavity as masses determined by electrospray mass spectroscopy, in the serving a function of Q/QH2 exchange was inferred from: (i) complex isolated from spinach (spinacea oleracea) the quinone analogue inhibitor, TDS, in both M. laminosus membranes [5]. In addition, a ninth subunit was found to be and C. reinhardtii, bound in an 11 Å×12 Å portal on the present in the complex from spinach which, at 35.3 kDa, is the p-side of the cavity [1,2]; (ii) electron density near the n-side largest in the set, and was identified to be ferredoxin:NADP+ of the cavity in M. laminosus most readily identified as reductase (FNR). The occupancy of FNR in the complex, where plastoquinone near the heme cn [1] (see section II.3). The it was first identified in biochemical studies [10,11], was other prosthetic groups observed to protrude or insert into the estimated to be 70–90% [11]. FNR has also been identified in Q/QH2 exchange cavity are: (i) the novel heme cn; (ii) the b6 f complex isolated from pea (H. Zhang and G. Kurisu, phytyl chain of the bound chlorophyll a [18,19] that extends unpublished). through the 11 Å×12 Å portal that connects the “exchange The dimeric complex is organized as a twenty-six trans- cavity” and the p-side quinone binding niche near the ISP membrane TM helix bundle, thirteen per monomer (view cluster [12–16]. In both b6 f and bc1 complexes, the portal parallel to membrane plane, Fig. 1), with the following defines the only route for QH2/Q entry/exit to the quinone distribution of TMH: cytochrome b6 (four), subunit IV binding niche near the ISP reaction site at the p-side interface (three), and cytochrome f, ISP, and the PetG, M, L, and N of the complex [1,2,20–22]. The penetration of the 20 carbon subunits (one each). As in the bc1 complex [12–16], the 25– phytyl chain through the portal results in partial occlusion of 27 residue TMH of the ISP is domain-swapped from the the portal, which would make the pathway of Q/QH2 transfer soluble domain in one monomer to the hydrophobic TM across the complex even more labyrinthine in the b6 f than in domain of the other (Fig. 1), a property that contributes to the bc1 complex. the stabilization of the dimer. 2.3. Heme x or ci or cn 2.2. The quinone exchange cavity; function of the dimer The structures of the cytochrome b6 f complex showed The two monomers enclose a large (30 Å along the the presence of a unique heme facing the inter-monomer symmetry axis of the dimer, 25 Å×15 Å in the membrane quinone exchange cavity on the n-side of the complex that plane (Fig. 1) cavity, the “quinone exchange cavity.” This is covalently linked by a single thioether bond to a Cys W.A. Cramer, H. Zhang / Biochimica et Biophysica Acta 1757 (2006) 339–345 341

2.5. Effects of inhibitors; visible and EPR spectral properties

The Em7 of heme cn is shifted by approximately−225 mV in the presence of the quinone analogue inhibitor, NQNO [28]. Heme cn is characterized by an absorbance band in redox difference spectra [28], or in CO-or butyl isocyanide- difference spectra [27], in the Soret region at approximately 425 nm. The pyridine hemochromagen redox difference spectrum for heme cn covalently bound to the cytochrome b polypeptide isolated from SDS-PAGE displays a broad spectrum of low amplitude in the α-band region with a peak at 553 nm, similar to that of other hemes with a single thioether linkage [31]. EPR spectra displaying ‘g’ values of the oxidized complex at 6.7 and 7.4 define heme cn as a high spin ferric heme in a rhombic environment [27]. In the crystal structures of the b6 f complex from both M. laminosus and C. reinhardtii, the binding site of heme cn (Fig. 2) occupies most of the binding site of the n-side quinone (Qn) and its analogue Fig. 2. Binding environment of hemes cn and heme b6. The color code is as inhibitor, , seen in the structure of the bc1 follows: b hemes (pink); the histidine ligands for heme (green); heme cn complex [12]. (orange); Cys35 (blue); and PQ (cyan). The water molecule bridging heme cn and heme bn is shown as a green ball. 2.6. Inferred function of heme cn; evolutionary origin

The absence of this heme in the bc1 complex implies that a residue (Cys35) on the electrochemically negative (n) side of likely function in the b6 f complex is -linked the cytochrome b6 polypeptide (Fig. 2). It is noted cyclic electron transport (section III.1). In addition, the elsewhere that, for purposes of teaching and inter-disciplin- sequence motifs coding for both hemes b and cn are seen not ary communication, this heme, which was initially called only in cyanobacteria, but also in the non-photosynthetic heme x and heme ci, respectively, in the structure from M. firmicutes that include gram-positive bacteria such as B. laminosus [1]and C. reinhardtii [2], is more appropriately subtilis (Table 1). The presence of the conserved residues denoted as heme cn [22]. The latter nomenclature incorpo- Cys35 that serves as a ligand for heme cn, and His86, 100, rates the facts that the heme is covalently bound, and 187, and 202, which serve as ligands for hemes bp and bn, can thereby c-type, as expressed in the notation originally used be noted. The presence of a complex resembling the in the C. reinhardtii structure [2], and that it is located on cytochrome b6 f complex [32] and of a covalently (-) the electrochemically negative (n-) side of the membrane. bound c-type heme [33] has been shown in B. subtilis, Before the determination of the X-ray structures, a heme of implying a metabolic role for these hemes in primitive non- unknown location, now identified as heme cn, was defined photosynthetic micro-. in two spectrophotometric studies, in which it was called “G” [23,24] and was shown to equilibrate in electron 3. Electron-proton transfer transfer with heme bn. This covalently bound heme in the b6 f complex was also observed in SDS-PAGE [25–28]. It has been inferred that electron transport and coupled proton translocation through the b6 f complex are described, + 2.4. Close proximity of hemes bn and cn: a di-heme complex? at least under conditions of low Δμ˜ H [34],bya“Q-cycle” model [35–37] essentially identical to that operating in the From the structure studies, heme cn is known to be located cytochrome bc1 complex from the respiratory chain and close (∼4 Å) to heme bn, which would allow rapid inter-heme purple photosynthetic bacteria [38–40]. Two experimental electron transfer. The Fe atom of heme cn is coordinated by a complications in establishing an unequivocal description of H2O or hydroxyl that is also H-bonded to the propionate of the the electron transfer pathway in the b6 f complex are: (i) the stromal side heme bn (Fig. 2). The midpoint potential of heme cn similarity (Δλmax ≅0.5 nm) of the peaks of the difference is pH-dependent in the physiological range, with an Em7 ≅ spectra of the two b hemes [24], which prevents direct +100 mV that is approximately 150 mV more positive than the measurement of inter-heme electron transfer; (ii) the absence potential of heme bn, for which the Em7 ≅−50 mV [29,30]. The of a quinone analogue inhibitor that binds tightly to a Qn proximity of hemes bn and cn raise the question of whether they site as does antimycin A in the bc1 complex. The effect of should be considered as two separate hemes or a single di-heme NQNO on the b6f complex mimics antimycin A in its cytochrome. In the latter case, the separation between the Em ability to block oxidation of heme bn [41,42], although this values of the two hemes would result partly from heme-heme does not result in a parallel inhibition of linear electron interactions. transport [43]. 342

Table 1 Sequence alignment of the 4_helix cytochome b polypeptide in the cytochrome bf (c) complexes from higher plant chloroplasts, green alga, cyanobacteria and eubacteria ..Cae,H hn iciiae ipyiaAt 77(06 339 (2006) 1757 Acta Biophysica et Biochimica / Zhang H. Cramer, W.A. – 345

Conserved Cys residues are highlighted in cyan. The two pairs of histidine ligands to the cytochrome b hemes are highlighted in red. (For interpretation of the references to colour in this table legend, the reader is referred to the web version of this article.) W.A. Cramer, H. Zhang / Biochimica et Biophysica Acta 1757 (2006) 339–345 343

The reactions of a Q-cycle mechanism that would describe the pathway of oxidation of plastoquinol through bifurcated high and low potential electron transfer pathways, are summarized (formulae 1–6): d PQH2 þ½2Fe 2S ðoxÞYPQþ½2Fe 2S ðredÞ þ 2Hþ ½pQside aqueous phaseð1Þ

FeS ðredÞþCyt f ðoxÞYFeS ðoxÞ þ Cyt f ðredÞ½high potential chainð2Þ

d PQþheme bp ðoxÞYPQ þ ð Þ½ ðÞ heme bp red low potential chain 3 Fig. 3. Proposed pathway, shown in one monomer of the b6f complex, of photosystem I and ferredoxinQmediated electron transport pathway that utilizes

bound FNR [10,11] and nQside hemes bn and cn reduce PQ located in the quinone heme bp ðredÞþheme bn ðoxÞYbp ðoxÞ exchange cavity (Fig. 1). The sequence of nQside electron transfer is based on [11, 28, 30 and Zhang and Cramer (unpublished data)]. þ bn ðredÞ½transQmembrane e transferð4Þ

heme bnðredÞþPQYheme bnðoxÞ Bassham C3 cycle requires three ATP and two NADPH per + þPQd½nQside etransferð5Þ CO2 fixed. The number of H needed to synthesize the required 3 ATP would then be 9–12, depending on the H+/ ATP stoichiometry, 3–4. In this case, a fully engaged Q d þ heme bnðredÞþPQþ2H YbnðoxÞ cycle would be sufficient to drive carbon fixation. (iii) From þ þ the perspective of the H+ requirement for rotation of the PQH2 ½nQside e transfer; H uptakeð6Þ ATPase, 3 ATP are synthesized in one complete rotation of the CF1 [45], the Fo sector of the ATPase contains 14 c- 3.1. Cyclic electron transport subunits [46], and thus 14 H+ are required for synthesis of the 3 ATP. In this case, H+ production by the linear electron A caveat to an exact application of the Q-cycle to the b6 f transfer pathway, even with a completely engaged Q cycle, complex of oxygenic photosynthesis is that the latter falls short of what is necessary to generate sufficient ATP for complex may also participate in a cyclic electron transport carbon fixation. In fact, ATP production by the linear pathway in which it acts as an electron acceptor from pathway is known to be less than fully efficient [47,48], ferredoxin or ferredoxin-FNR that is reduced by the implying that it is necessary to produce ATP by the PSI- photosystem I reaction center (Fig. 3). Thus, the n-side of linked cyclic pathway. the b6 f complex would participate in electron transfer The role of the b6 f complex in this cyclic pathway has been reactions with a second electron donor-acceptor system. extensively debated. On one hand, a comparison of the effect of The ATP provided by such a cyclic electron transport antimycin A on flash-induced reduction of cytochrome b heme + pathway, coupled to formation of Δμ˜ H, is a metabolic and ferredoxin-dependent ATP synthesis led to the conclusion requirement to establish a proper balance of NADPH and that the b6 f complex is not involved in the cyclic pathway [49– ATP for the fixation of CO2 [44]. The magnitude of the 51]. In retrospect, the effects of antimycin A on cytochrome b contribution of this cyclic pathway to the generation of the reduction have been ambiguous [49,50], and it is now known + Δμ˜ H and synthesis of ATP is not precisely known, and the that the binding site of antimycin A in the bc1 complex is regulatory mechanisms that may apply under different light occluded by heme cn. NADH dehydrogenase (NDH) can intensities and conditions of plant stress are not well provide an alternative pathway for cyclic electron transport understood. However, the requirement of the cyclic pathway [52,53]. However, relative to the b6 f complex, the amount of and the extent of its contribution can be estimated from ndh that can be isolated from any oxygenic photosynthetic + + information on the utilization of the Δμ˜ H by the H -ATPase, membrane system is small. Recent studies indicate kinetic as follows: (i) The maximum H+/2e− ratio =6 for a two competence of a ferredoxin/FNR cyclic pathway through the + + − electron reduction of NADP , and H /4e ratio=12 for b6 f complex that operates most efficiently in dark-adapted reduction of two NADP+, via linear electron transport from leaves or when photosystem II is inhibited, and which resides in water, assuming that a “Q cycle” is completely obligatory the structurally separated non-appressed region of the thylakoid and continuously engaged [37]. In this mechanism, 2/3 of membrane [54–56]. + the protons that form the Δμ˜ H result from electron transfer A role of heme cn in an n-side ferredoxin-and FNR- through the b6 f complex. The remainder of the proton dependent pathway is implied by: (i) the ferredoxin [30,57] deposition on the p-side of the membrane results from water and FNR [11] dependence of heme b reduction by NADPH; oxidation by PSII. (ii) Carbon fixation by the Calvin- (ii) a ΔEm between hemes bn and cn [28] that implies that the 344 W.A. Cramer, H. Zhang / Biochimica et Biophysica Acta 1757 (2006) 339–345

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