G. M. Soriano

Comparison of the cytochrome bc1 complex with the anticipated structure of the cytochrome b6f complex: Le plus ça change le plus c'est la même chose.

Structural alignment of the integral cytochrome b6-SU IV subunits with the solved structure of themitochondrial bc1 complex shows a pronounced asymmetry. There is a much higher homology onthe p-side of the membrane, suggesting a similarity in the mechanisms of intramembrane andinterfacial electron and proton transfer on the p-side, but not necessarily on the n-side. Structuraldifferences between the bc1 and b6f complexes appear to be larger the farther the domain or subunitis removed from the membrane core, with extreme differences between cytochromes c1 and f. Aspecial role for the dimer may involve electron sharing between the two hemes bp, which is indicatedas a probable event by calculations of relative rate constants for intramonomer heme bp → hemebn, or intermonomer heme bp → heme bp electron transfer. The long-standing observation offlash-induced oxidation of only ∼0.5 of the chemical content of cyt f may be partly a consequence ofthe statistical population of ISP bound to cyt f on the dimer. It is proposed that the p-side domainof cyt f is positioned with its long axis parallel to the membrane surface in order to: (i) allow itslarge and small domains to carry out the functions of cyt c1 and suVIII, respectively, of the bc1complex, and (ii) provide maximum dielectric continuity with the membrane. (iii) This positionwould also allow the internal water chain (“proton wire”) of cyt f to serve as the p-side exit portfor an intramembrane H+ transfer chain that would deprotonate the semiquinol located in themyxothiazol/MOA-stilbene pocket near heme bp. A hypothesis is presented for the identity of theamino acid residues in this chain.

Electron transfer from the Rieske iron-sulfur protein (ISP) to cytochrome f in vitro. Is a guided trajectory of the ISP necessary for competent docking?

The time course of electron transfer in vitro between soluble domains of the Rieske iron-sulfur protein (ISP) and cytochrome f subunits of the cytochromeb 6 f complex of oxygenic photosynthesis was measured by stopped-flow mixing. The domains were derived from Chlamydomonas reinhardtii and expressed inEscherichia coli. The expressed 142-residue soluble ISP apoprotein was reconstituted with the [2Fe-2S] cluster. The second-order rate constant,k 2 ( ISP f ) = 1.5 × 106 m −1s−1 , for ISP to cytochrome felectron transfer was <10−2 of the rate constant at low ionic strength,k 2 ( f PC ) (> 200 × 106 m −1 s−1), for the reduction of plastocyanin by cytochrome f, and ∼1/30 ofk 2 ( f PC ) at the ionic strength estimated for the thylakoid interior. In contrast tok 2 ( f PC ) ,k 2 ( ISP f ) was independent of pH and ionic strength, implying no significant role of electrostatic interactions. Effective pK values of 6.2 and 8.3, respectively, of oxidized and reduced ISP were derived from the pH dependence of the amplitude of cytochrome f reduction. The first-order rate constant,k 1 ( ISP f ) , predicted fromk 2 ( ISP f ) is ∼10 and ∼150 times smaller than the millisecond and microsecond phases of cytochrome f reduction observed in vivo. It is proposed that in the absence of electrostatic guidance, a productive docking geometry for fast electron transfer is imposed by the guided trajectory of the ISP extrinsic domain. The requirement of a specific electrically neutral docking configuration for ISP electron transfer is consistent with structure data for the related cytochromebc 1 complex.

Characterization and Crystallization of A Highly Active Cytochrome b 6 f Complex from the Thermophilic Cyanobacterium, Mastigocladus Laminosus

The cytochrome b6f complex, one of four integral protein complexes in the oxygenic photosynthetic membrane, mediates electron transport, H+ translocation, and generation of the electrochemical proton gradient (1–3), and may also participate in other non-bioenergetic functions, such as activation of a redox-controlled protein kinase (4). The polypeptide content of the b6f complex, purified from spinach chloroplasts (5) or the green alga, C. reinhardtii (6), includes 4 proteins of Mr > 17,000, all of which contain redox prosthetic groups (cytochromesf, b6, Rieske protein) and/or participate in quinone binding: the petA gene product, cytochrome f (MW = 32,038 in spinach chloroplasts); petB, cytochrome b6 (MW = 24,166); the petC Rieske [2Fe-2S] protein (MW = 19,116); and subunit IV (MW = 17,445). The complex also contains 3 small chloroplast-encoded hydrophobic subunits, petG (7), petM (8), and petL (9) consisting of 37, 39, and 32 residues. The molecular weight of the monomeric complex of 7 polypeptide components with unity stoichiometry, together with prosthetic groups, is approximately 110 kDa. The number of trans-membrane a-helices in the monomeric complex, inferred from nucleotide sequences and biochemical topography and binding studies, is 12 (4 in PetB, 3 in PetD, 1 each in PetA, C, G, L, and M). A dimeric b6f complex (10), would include 24 trans-membrane α-helices and have a molecular weight ≈ 220 kDa.

Identification of the Basic Residues of Cytochrome F Responsible for Electrostatic Docking with Plastocyanin in Vitro : Relevance to the Electron Transfer in Vivo

Cytochrome f is the largest polypeptide of the b 6 f complex of oxygenic photosynthesis (1). The b 6 f complex mediates electron transfer between Photosystems I and II, and translocates protons across the thylakoid membrane (2). One of the interesting features of the x-ray structure of the redox-active soluble fragment of cytochrome f (cyt f) is the presence of a dominant patch of basic Lys residues near the interface of the small and large domains (3). On the other hand, the crystal structure of its physiological electron acceptor, plastocyanin (4,5), revealed the presence of a negative patch around Tyr83. These two sites of localized complementary charges in the two proteins have been proposed to participate in the docking reactions of cyt f and PC (3). The two proteins are shown in a “pre-docking” configuration in (6). Comparison of amino acid sequences across many plant and algal species showed a high degree of conservation of the residues that are in the positive and negative patches of cyt f and plastocyanin (PC), respectively (7). These findings, in addition to: (i) the observed decrease in the rate of cyt f oxidation by PC with increasing ionic strength (8-11), (ii) chemical cross-linking of cyt f Lys 187 to PC Asp44 (12), and (iii) computer simulations of cyt f /PC docking (13,14), argue strongly for a dominant role of electrostatic interactions in cyt f /PC reaction.