Reaction of cytochrome c and c2 with the Rhodobacter sphaeroides reaction center involves the heme crevice domain

589

329

The three-dimensional structure of the protein subunits of the reaction center (RC) of Rhodobacter sphaeroides has been determined by x-ray diffraction at a resolution of 2.8 A with an R factor of 26%. The L and M subunits each contain five transmembrane helices and several helices that do not span the membrane. The L and M subunits are related to each other by a 2-fold rotational symmetry axis that is approximately the same as that determined for the cofactors. The H subunit has one transmembrane helix and a globular domain on the cytoplasmic side, which contains a helix that does not span the membrane and several 13-sheets. The structural homology with RCs from other purple bacteria is discussed. A structure of the complex formed between the water soluble cytochrome c2 and the RC from Rb. sphaeroides is proposed.The reaction center (RC) from photosynthetic bacteria is an integral membrane protein complex, which contains a number of cofactors that mediate the primary photochemistry. The RC of Rhodobaccter sphaeroides is composed of three protein subunits L, M, and H having 281, 307, and 260 residues, respectively (1-3). From an analysis of the amino acid sequence of the subunits, five membrane-spanning helices in both the L and M subunits and one in the H subunit were predicted (1-3). Furthermore, labeling experiments showed that the RC spans the membrane with the bulk of the H subunit on the cytoplasmic side; the amino terminus of L was determined to be on the cytoplasmic side (for reviews, see refs. 4 and 5).The three-dimensional structure of the RC has been obtained to a resolution of 2.8 A. In a previous paper, we described the x-ray diffraction analysis and reported the structure of the cofactors (6). In this paper, we present the structure of the individual subunits as well as the entire RC complex. A structure of the complex between the RC and the water soluble cytochrome c, is proposed. The overall structure and homology with other RCs, in particular that of Rhodopseudomonas viridis (7), is discussed. METHODS RC crystals having the space group P212121 were analyzed by x-ray diffraction as described (6). The structure ofthe RC was determined at a resolution of 2.8 A with an R factor of 26% between calculated and observed structure factors. The secondary structure was identified on the basis of main-chain hydrogen bonding and torsion angle patterns. Due to deviations from ideal geometry, the boundaries of the regions of a-helices and a-sheets have an uncertainty of several residues. Final assignments of the boundaries were checked visually by computer graphics [Evans and Sutherland PS 300 with FRODO program (8)].The directions of the helical axes were equated with the normals of the greatest square plane fit of the Ca atoms of each helix (9). The radius of curvature to an a-helix was calculated by two methods: (i) from the radius of the best circle fit to the projections of Ca atoms onto the helical axis (10); and (ii) from the directions of the helical axes based on residues in the first and last...

supporting

confidence: 89%

“…The binding domain on the RC contains negatively charged carboxylate groups (19) that are believed to interact electrostatically (for a review, see ref. 20), with lysine residues surrounding the heme crevice of cytochrome C2 (21).…”

mentioning

confidence: 99%

Structure of the reaction center from Rhodobacter sphaeroides R-26: the protein subunits.

Allen

Fehér²,

Yeates³

et al. 1987

589

329

“…Changes to charge-surface groups near the binding interface by using chemical modification (22,23) and site-directed mutagenesis (13,24,25) were shown to change the association rate. The role of electrostatic interactions in protein association was demonstrated by modeling studies (26)(27)(28)(29)(30).…”

Section: [1]mentioning

confidence: 99%

Transition state and encounter complex for fast association of cytochrome c ₂ with bacterial reaction center

Miyashita

Onuchic

Okamura

2004

Electrostatic interactions strongly enhance the electron transfer reaction between cytochrome (Cyt) c 2 and reaction center (RC) from photosynthetic bacteria, yielding a second-order rate constant, k 2 Ϸ 10 9 s ؊1 ⅐M ؊1 , close to the diffusion limit. The proposed mechanism involves an encounter complex (EC) stabilized by electrostatic interactions, followed by a transition state (TS), leading to the bound complex active in electron transfer. The effect of electrostatic interactions was previously studied by Tetreault et al. electron transfer ͉ photosynthesis ͉ electrostatics ͉ site-directed mutagenesis ͉ correlation I ntermolecular electron-transfer reactions play important roles in many biological processes such as photosynthesis and respiration (1). In these reactions, two electron-transfer proteins associate to form an electron donor-acceptor complex in which electron transfer occurs (2). Electrostatic interactions can greatly enhance the rate of protein association by providing long-range guidance in the association process (3). For fast processes assisted by electrostatic interactions, a two-step association mechanism has been proposed (4, 5). The first step is the formation of a loosely bound encounter complex (EC), followed by a rate-limiting process through a transition state (TS), leading to the bound active state. In this paper we examine the association process for the reaction center (RC) and cytochrome (Cyt) c 2 that are involved in the electron-transfer chain in bacterial photosynthesis by using electrostatic calculations and experimental data to obtain a molecular description of the TS and the EC. The results of these calculations describe the ensemble of states that represent a roadmap for the association process and the interactions likely to be important in the dynamics. This approach is complementary to Brownian dynamics simulations (5-7) that yield the diffusional rate constant.The RC is the membrane protein involved in the primary light-induced electron-transfer step in bacterial photosynthesis (8,9). Light absorbed by RC pigments activates photooxidation of the primary donor, D, a bacteriochlorophyll dimer, leading to the reduction of a quinone molecule Q, DQ 3 D ϩ Q Ϫ . The electron from Q Ϫ cycles back to D ϩ through an electron-transfer chain involving the membrane-bound Cyt bc1 complex. The last step in the cycle involves a soluble Cyt c 2 molecule that shuttles the electron back to the RC to complete the cyclic electron-transfer process. This cyclic electron transfer is coupled to proton pumping across the membrane that drives ATP synthesis.The rates of electron transfer between isolated RCs and Cyt c 2 have been measured by using laser-flash techniques and shown to be well optimized for cyclic electron transfer by using the scheme below (refs. 10-13; reviewed in ref. 14).[1]Before the laser flash, the Cyt and RC (denoted DQ) are in equilibrium between a bound state and a free state with the dissociation constant K d Ϸ 10 Ϫ6 M at low ionic strength (Ϸ5 mM) (13). After a laser flash, t...

“…The effect is quantitatively similar to that obtained with cytochrome c2 in either Rb. sphaeroides (47) protein-protein interactions (48), can be applied. A fit of the experimental data to the following equation (48) e-Kp lnk = lnk,.-Viil + Kp, [2] in which K …”

mentioning

confidence: 99%

Kinetics of photo-induced electron transfer from high-potential iron-sulfur protein to the photosynthetic reaction center of the purple phototroph Rhodoferax fermentans.

Hochkoeppler

Zannoni

Ciurli

et al. 1996

The kinetics of photo-induced electron transfer from high-potential iron-sulfur protein (HiPIP) to the photosynthetic reaction center (RC) of the purple phototroph Rhodoferaxfermentans were studied. The rapid photooxidation of heme c-556 belonging to RC is followed, in the presence of HiPIP, by a slower reduction having a second-order rate constant of4.8 x 107 M -l s -1. The limiting value of k.bs at high HiPIP concentration is 95 s-1. The amplitude of this slow process decreases with increasing HiPIP concentration. The amplitude of a faster phase, observed at 556 and 425 nm and involving heme c-556 reduction, increases proportionately. The rate constant of this fast phase, determined at 425 and 556 nm, is -3 x 105 s-1. This value is not dependent on HiPIP concentration, indicating that it is related to a first-order process. These observations are interpreted as evidence for the formation of a HiPIP-RC complex prior to the excitation flash, having a dissociation constant of -2.5 ,uM. The fast phase is absent at high ionic strength, indicating that the complex involves mainly electrostatic interactions. The ionic strength dependence of kobs for the slow phase yields a second-order rate constant at infinite ionic strength of 5.4 x 106 M-t.s-I and an electrostatic interaction energy of -2.1 kcal/mol (1 cal = 4.184 J). We conclude that Rhodoferax fermentans HiPIP is a very effective electron donor to the photosynthetic RC.Anoxygenic phototrophic bacteria contain two membranebound components that are essential for energy production, the photosynthetic reaction center (RC) and the cytochrome bc1 complex. Two types of RC have been structurally characterized: the RC from Rhodobacter sphaeroides is made of three subunits (1, 2), while, in addition to these, RC from Rhodopseudomonas viridis contains a fourth tetraheme cytochrome c subunit (2, 3). InRps. viridis the four heme groups have a bands at 559, 552, 556, and 554 nm, and reduction potentials of +380 mV, +20 mV, +310 mV, and -60 mV, respectively (4). The four hemes are arranged in an almost linear fashion, (2,5,6) and are aligned in the sequence P/c-559/c-552/c-556/c-554, where P indicates the bacteriochlorophyll special pair (4, 7). The highest potential heme (c-559) is oxidized the most rapidly (t/2 -300 ns) by the photooxidized P (P+), followed by a slower oxidation of heme c-556 (ti/2 2 ,us) (4,8,9). The quinol generated by RC photooxidation is utilized by the bc1 complex to reduce cytochrome c2 (10), a soluble protein closely related to mitochondrial cytochrome c (11,12). Cytochrome c2 then reduces the photooxidized RC, closing the photocycle. However, although cytochrome c2 reacts directly with P+ in Rb. sphaeroides (13-21), in Rps. viridis, it reacts with the tetraheme subunit (22-24). Only about half of the purple bacterial species described so far contain cytochrome c2 (25), and it is not known how the remaining species carry out electron transfer between the bc1 and RC complexes.The RC from the purple nonsulfur bacterium Rhodoferax fermentans (26)...