Continuum electrostatic model for the binding of cytochrome c2 to the photosynthetic reaction center from Rhodobacter sphaeroides

Electrostatic interactions are important for protein-protein association. In this study, we examined the electrostatic interactions between two proteins, cytochrome c(2) (cyt c(2)) and the reaction center (RC) from the photosynthetic bacterium Rhodobacter sphaeroides, that function in intermolecular electron transfer in photosynthesis. Electrostatic contributions to the binding energy for the cyt c(2)-RC complex were calculated using continuum electrostatic methods based on the recent cocrystal structure [Axelrod, H. L., et al. (2002) J. Mol. Biol. 319, 501-515]. Calculated changes in binding energy due to mutations of charged interface residues agreed with experimental results for a protein dielectric constant epsilon(in) of 10. However, the electrostatic contribution to the binding energy for the complex was close to zero due to unfavorable desolvation energies that compensate for the favorable Coulomb attraction. The electrostatic energy calculated as a function of displacement of the cyt c(2) from the bound position showed a shallow minimum at a position near but displaced from the cocrystal configuration. These results show that although electrostatic steering is present, other short-range interactions must be present to contribute to the binding energy and to determine the structure of the complex. Calculations made to model the experimental data on association rates indicate a solvent-separated transition state for binding in which the cyt c(2) is displaced approximately 8 A above its position in the bound complex. These results are consistent with a two-step model for protein association: electrostatic docking of the cyt c(2) followed by desolvation to form short-range van der Waals contacts for rapid electron transfer.     

Unfolding pathway and intermolecular interactions of the cytochrome subunit in the bacterial photosynthetic reaction center

Precise folding of photosynthetic proteins and organization of multicomponent assemblies to form functional entities are fundamental to efficient photosynthetic electron transfer. The bacteriochlorophyll b-producing purple bacterium Blastochloris viridis possesses a simplified photosynthetic apparatus. The light-harvesting (LH) antenna complex surrounds the photosynthetic reaction center (RC) to form the RC-LH1 complex. A non-membranous tetraheme cytochrome (4Hcyt) subunit is anchored at the periplasmic surface of the RC, functioning as the electron donor to transfer electrons from mobile electron carriers to the RC. Here, we use atomic force microscopy (AFM) and single-molecule force spectroscopy (SMFS) to probe the long-range organization of the photosynthetic apparatus from Blc. viridis and the unfolding pathway of the 4Hcyt subunit in its native supramolecular assembly with its functional partners. AFM images reveal that the RC-LH1 complexes are densely organized in the photosynthetic membranes, with restricted lateral protein diffusion. Unfolding of the 4Hcyt subunit represents a multi-step process and the unfolding forces of the 4Hcyt α-helices are approximately 121 picoNewtons. Pulling of 4Hcyt could also result in the unfolding of the RC L subunit that binds with the N-terminus of 4Hcyt, suggesting strong interactions between RC subunits. This study provides new insights into the protein folding and interactions of photosynthetic multicomponent complexes, which are essential for their structural and functional integrity to conduct photosynthetic electron flow.     

Binding of cytochrome c2 to the isolated reaction center of Rhodospirillum rubrum involves the "backside" of cytochrome c2

Lys 109, Lys 112 and Glu 1 of cytochrome c2 from Rhodospirillum rubrum G-9 are about 4-fold less reactive towards acetic anhydride when cytochrome c2 is bound to the isolated photosynthetic reaction center from the same organism. The three shielded residues are clustered together on the "backside" of cytochrome c2. This contrasts with mitochondrial cytochrome c where "frontside" lysines are protected by different physiological electron transfer partners.     

Comparison of the binding sites for high-potential iron-sulfur protein and cytochrome c on the tetraheme cytochrome subunit bound to the bacterial photosynthetic reaction center

A tetraheme cytochrome subunit bound to the photosynthetic reaction center (RC) of purple bacterium, Rubrivivax gelatinosus, interacts with two types of soluble electron donors, cytochromes c and high-potential iron-sulfur protein (HiPIP), at a binding domain in the vicinity of low-potential heme 1, the fourth heme from the special pair of bacteriochlorophyll. To clarify the mechanism of the interaction, the domain around heme 1 was examined using site-directed mutants that changed the surface charge in the region within 20 A from the heme edge. In the case of the interaction with soluble cytochrome c, a strong dependence on the sign of the introduced charge was observed in all mutants: positive charge inhibited the reaction rate, whereas additional negative charge accelerated it. This confirmed the electrostatic nature of the binding. Interaction with HiPIP was inhibited by a limited number of mutations at the close vicinity of heme 1, and no acceleration was observed (the effects of some mutations were independent of the sign of the introduced charge). The acidic residues which were critically important for the binding of cytochrome c showed much less contribution to the binding of HiPIP. The binding site for HiPIP appears to be mostly formed by uncharged and hydrophobic residues, occupying a significantly smaller area than the cytochrome-c-binding site. It is proposed that the docking of HiPIP to the RC in Rvi. gelatinosus is primarily controlled by hydrophobic contacts between protein surfaces, thus differing from the electrostatic mode of the RC-cytochrome c interaction.     

The use of photoactivated hetero-bifunctional reagents to cross-link cytochrome c to proteins that bind it by electrostatic interactions.

Lysine residues of horse heart cytochrome c have been modified with N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS) and ethyl N-5-azido-2-nitrobenzoylaminoacetimidate (ANB-AI), reagents that attach nitroaryl azides onto the surface of proteins by amide and amidine linkages, respectively. When acting as an electron acceptor for yeast cytochrome b₂, modification of cytochrome c with ANB-NOS increases the K_m for the reaction by 2-fold, while modification with ANB-AI has little effect on the K_m. The V_max for the reduction of cytochrome c by cytochrome b₂ is reduced by the attachment of both compounds to cytochrome c. When the modified cytochromes c were illuminated with phosvitin, cytochrome b₅, and cytochrome c peroxidase, cross-linked species were formed which could be resolved by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. In each case the amidine derivatives of cytochrome c modified with ANB-AI showed more cross-linking than the amide derivatives of cytochrome c modified with ANB-NOS. When the modified cytochromes c were present in a 3-fold excess of phosvitin, cross-linked products containing 1, 2, and 3 molecules of cytochrome c covalently attached to phosvitin were observed. Photolysis of the modified cytochromes c in the presence of cytochrome b₅, resulted in the formation of a cross-linked 1:1 complex between the two cytochromes as well as higher order aggregates containing up to 5 molecules of cytochrome c plus cytochrome b₂. When cytochrome c peroxidase was illuminated with the modified cytochromes c, the predominant cross-linked product was a 1:1 complex between the two heme proteins. However, a cross-linked species was detected in small amounts with the apparent composition of 2 molecules of cytochrome c and 1 of the peroxidase. Also, a procedure is described for the synthesis of ANB-AI with ¹⁴C in the imidocarbon which is ultimately derived from ¹⁴CN.     

Characterisation of ionisations that influence the redox potential of mitochondrial cytochrome c and photosynthetic bacterial cytochromes c2

Several cytochromes c₂ from the Rhodospirillaceae show a pH dependence of redox potential in the physiological pH range which can be described by equations involving an ionisation in the oxidised form (pK_o) and one in the reduced form (pK_r). These cytochromes fall into one of two groups according to the degree of separation of pK_o and pK_r. In group A, represented here by the Rhodomicrobium vannielii cytochrome c₂, the separation is approx. one pH unit and the ionisation is that of a haem propionic acid. Members of this group are unique among both cytochromes c₂ and mitochondrial cytochromes c in lacking the conserved residue Arg-38. We propose that the role of Arg-38 is to lower the pK of the nearby propionic acid, so that it lies out of the physiological pH range. Substitution of this residue by an uncharged amino acid leads to a raised pK for the propionic acid. In group B, represented here by Rhodopseudomonas viridis cytochrome c₂, the separation between pK_o and pK_r is approx. 0.4 pH unit and the ionisable group is a histidine at position 39. This was established by NMR spectroscopy and confirmed by chemical modification. Only a few other members of the cytochrome c₂/mitochondrial cytochrome c family have a histidine at this position and of these, both Crithidia cytochrome c-557 and yeast cytochrome c were found to have a pH-dependent redox potential similar to that of Rps. viridis cytochrome c₂. Using Coulomb's law, it was found that the energy required to separate pK_o and pK_r could be accounted for by simple electrostatic interactions between the haem iron and the ionisable group.     

Kinetic studies on the reaction between cytochrome c oxidase and ferrocytochrome c.

In stopped-flow experiments in which oxidized cytochrome c oxidase was mixed with ferrocytochrome c in the presence of a range of oxygen concentrations and in the absence and presence of cyanide, a fast phase, reflecting a rapid approach to an equilibrium, was observed. Within this phase, one or two molecules of ferrocytochrome were oxidized per haem group of cytochrome a, depending on the concentration of ferrocytochrome c used. The reasons for this are discussed in terms of a mechanism in which all electrons enter through cytochrome a, which, in turn, is in rapid equilibrium with a second site, identified with 'visible' copper (830 nm-absorbing) Cud (Beinert et al., 1971). The value of the bimolecular rate constant for the reaction between cytochromes c2+ and a3+ was between 10(6) and 10(7) M(-1)-S(-1); some variability from preparation to preparation was observed. At high ferrocytochrome c concentrations, the initial reaction of cytochrome c2+ with cytochrome a3+ could be isolated from the reaction involving the 'visible' copper and the stoicheiometry was found to approach one molecule of cytochrome c2+ oxidized for each molecule of cytochrome a3+ reduced. At low ferrocytochrome c concentrations, however, both sites (i.e. cytochrome a and Cud) were reduced simultaneously and the stoicheiometry of the initial reaction was closer to two molecules of cytochrome c2+ oxidized per molecule of cytochrome a reduced. The bleaching of the 830 nm band lagged behind or was simultaneous with the formation of the 605 nm band and does not depend on the cytochrome c concentration, whereas the extinction at the steady-state does. The time-course of the return of the 830 nm-absorbing species is much faster than the bleaching of the 605 nm-absorbing component, and parallels that of the turnover phase of cytochrome c2+ oxidation. Additions of cyanide to the oxidase preparations had no effect on the observed stoicheiometry or kinetics of the reduction of cytochrome a and 'visible' copper, but inhibited electron transfer to the other two sites, cytochrome a3 and the undetectable copper, Cuu.     

Identification of the cystic fibrosis transmembrane conductance regulator domains that are important for interactions with ROMK2

In addition to functioning as a cAMP-activated chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR) plays an important role in conferring regulatory properties on other ion channels. It is known, with respect to CFTR regulation of ROMK2 (renally derived K(ATP) channel), that the first transmembrane domain and the first nucleotide binding fold domain (NBF1) of CFTR are necessary for this interaction to occur. It has been shown that under conditions that promote phosphorylation, the ROMK2-CFTR interaction is attenuated. To elucidate the complex nature of this interaction, CFTR constructs were co-expressed with ROMK2 in Xenopus oocytes, and two microelectrode voltage clamp experiments were performed. Although the second half of CFTR can act as a functional chloride channel, our results suggest that it does not confer glibenclamide sensitivity on ROMK2, as does the first half of CFTR. The attenuation of the ROMK2-CFTR interaction under conditions that promote phosphorylation is dependent on at least the presence of the R domain of CFTR. We conclude that transmembrane domain 1, NBF1, and the R domain are the CFTR domains involved in the ROMK2-CFTR interaction and that NBF2 and transmembrane domain 2 are not essential. Lastly, the R domain of CFTR is necessary for the attenuation of the ROMK2-CFTR interaction under conditions that promote phosphorylation.     

Lysines in the amino-terminal alpha-helix are important to the stability of Rhodobacter capsulatus cytochrome c2.

The protein stabilities of wild type and four site-directed mutants of Rhodobacter capsulatus cytochrome c2 have been characterized. The integrity of the cytochrome c2 iron-sulfur environment was ascertained by titration of the 696-nm absorbance band with alkali, and the conformational stability was determined by titration of the 220-nm circular dichroism signal with Gdn-HCl. Analysis of the alkaline transition pK value of K12D (lysine-12 substituted by aspartate) indicated that the K12D iron-sulfur environment was destabilized by 0.6 kcal/mol relative to the wild-type cytochrome c2 at low ionic strength. In contrast, the alkaline transition pK values of K14E (lysine-14 substituted by glutamate), K32E (lysine-32 substituted by glutamate), and K14E/K32E (lysines-14 and -32 substituted by glutamates) were indistinguishable from the wild type, indicating that these substitutions have no effect on the stability of the iron-sulfur environment. Gdn-HCl denaturation of K12D and K14E indicated that both these mutations decreased conformational stability by 1.3 kcal/mol. In contrast, mutant K32E exhibited a small stabilizing effect of 0.2 kcal/mol. Gdn-HCl denaturation of K14E/K32E indicated that this mutation decreased conformational stability by 1.3 kcal/mol, which is consistent with the additive effects of the single charge mutations at positions 14 and 32. The conformational instability of mutants possessing negative charges at position 12 or 14 is best explained by their positioning at the carboxy-terminal region of the amino-terminal alpha-helix of R. capsulatus cytochrome c2. Accordingly, introduction of negatively charged groups into this region appears to destabilize cytochrome c2 through energetically unfavorable interactions with the dipole of the amino-terminal helix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions between cytochrome c2 and the photosynthetic reaction center from Rhodobacter sphaeroides: the cation-pi interaction

The cation-pi interaction between positively charged and aromatic groups is a common feature of many proteins and protein complexes. The structure of the complex between cytochrome c(2) (cyt c(2)) and the photosynthetic reaction center (RC) from Rhodobacter sphaeroides exhibits a cation-pi complex formed between Arg-C32 on cyt c(2) and Tyr-M295 on the RC [Axelrod, H. L., et al. (2002) J. Mol. Biol. 319, 501-515]. The importance of the cation-pi interaction for binding and electron transfer was studied by mutating Tyr-M295 and Arg-C32. The first- and second-order rates for electron transfer were not affected by mutating Tyr-M295 to Ala, indicating that the cation-pi complex does not greatly affect the association process or structure of the state active in electron transfer. The dissociation constant K(D) showed a greater increase when Try-M295 was replaced with nonaromatic Ala (3-fold) as opposed to aromatic Phe (1.2-fold), which is characteristic of a cation-pi interaction. Replacement of Arg-C32 with Ala increased K(D) (80-fold) largely due to removal of electrostatic interactions with negatively charged residues on the RC. Replacement with Lys increased K(D) (6-fold), indicating that Lys does not form a cation-pi complex. This specificity for Arg may be due to a solvation effect. Double mutant analysis indicates an interaction energy between Tyr-M295 and Arg-C32 of approximately -24 meV (-0.6 kcal/mol). This energy is surprisingly small considering the widespread occurrence of cation-pi complexes and may be due to the tradeoff between the favorable cation-pi binding energy and the unfavorable desolvation energy needed to bury Arg-C32 in the short-range contact region between the two proteins.     

Studies of sperm from mutant mice suggesting that two neurotransmitter receptors are important to the zona pellucida-initiated acrosome reaction

Two sperm neurotransmitter receptor/channels, the glycine receptor (GlyR) and a nicotinic acetylcholine receptor containing an alpha7 subunit (alpha7nAChR) were previously shown to be important to the mouse acrosome reaction (AR) initiated by solubilized egg zona pellucida (ZP). Here, we investigated whether sperm from homozygous mutant mice with a single amino acid mutation in the alpha subunit of their GlyR and sperm from homozygous mutant mice with an engineered disruption of the gene for the nicotinic acetylcholine receptor alpha7 subunit could undergo the AR on ZP-intact eggs. Wild-type and mutant sperm were treated with 3-quinuclidinyl benzilate (QNB), known to be an inhibitor of the ZP-initiated AR (but shown in the present work not to inhibit the acetylcholine-initiated AR). The ZP-initiated AR on ZP-intact eggs should occur only in sperm not treated with QNB. The absence of such an increase in the untreated mutant sperm would demonstrate that such sperm were unable to respond to the intact ZP. The results demonstrated for the first time that GlyR mutant sperm do not undergo the AR on ZP-intact mouse eggs, and that their ability to fertilize is inhibited by 63% in vitro. Moreover, we found that GlyR mutant sperm exhibited normal capacitation and confirmed that they not undergo the AR initiated by solubilized mouse ZP. Our studies demonstrated for the first time that sperm from mutant alpha7nAChR mice exhibit normal capacitation, do not undergo the AR in response to acetylcholine, solubilized ZP or on ZP-intact eggs, and display a 25% reduction in fertilization in vitro. This is the first genetic evidence for the importance of the alpha7nAChR in the ZP-initiated AR. While defects in either the GlyR or the alpha7nAChR completely inhibit the ZP-initiated AR, fertilization by these mutant sperm can still occur in vitro, probably due to sperm that complete spontaneous AR on the ZP.     

The acidic nature of the CcmG redox-active center is important for cytochrome c maturation in Escherichia coli

Cytochrome c biogenesis in Escherichia coli is a complex process requiring at least eight genes (ccmABCDEFGH). One of these genes, ccmG, encodes a thioredoxin-like protein with unusually specific redox activity. Here, we investigate the basis for CcmG function and demonstrate the importance of acidic residues surrounding the redox-active center.     

Spectroscopic studies of bound cytochrome c and an iron-sulfur center in a purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Flash-induced optical kinetics at room temperature of cytochrome (Cyt) c ₅₅₁ and an Fe-S center (CF_A/CF_B) bound to a purified reaction center (RC) complex from the green sulfur photosynthetic bacterium Chlorobium tepidum were studied. At 551 nm, the flash-induced absorbance change decayed with a t _1/2 of several hundred ms, and the decay was accelerated by 1-methoxy-5-methylphenazinium methyl sulfate (mPMS). In the blue region, the absorbance change was composed of mPMS-dependent (Cyt) and mPMS-independent component (CF_A/CF_B) which decayed with a t _1/2 of 400–650 ms. Decay of the latter was effectively accelerated by benzyl viologen (Em –360 mV) and methyl viologen (–440 mV), and less effectively by triquat (–540 mV). The difference spectrum of Cyt c had negative peaks at 551, 520 and 420 nm, with a positive rise at 440 to 500 nm. The difference spectrum of CF_A/CF_B resembled P430 of PSI, and had a broad negative peak at 430435 nm.Abbreviations (B)Chl (bacterio)chlorophyll - Cyt cytochrome - F_A, F_B and F_X iron-sulfur center A, B and X of Photosystem I - CF_A, CF_B and CF_X F_A-,F_B- and F_X-like Fe-S center of Chlorobium - mPMS 1-methoxy-5-methylphenazinium methyl sulfate - PSI Photosystem I - RC reaction center     

X-ray structure determination of the cytochrome c2: reaction center electron transfer complex from Rhodobacter sphaeroides

In the photosynthetic bacterium Rhodobacter sphaeroides, a water soluble cytochrome c2 (cyt c2) is the electron donor to the reaction center (RC), the membrane-bound pigment-protein complex that is the site of the primary light-induced electron transfer. To determine the interactions important for docking and electron transfer within the transiently bound complex of the two proteins, RC and cyt c2 were co-crystallized in two monoclinic crystal forms. Cyt c2 reduces the photo-oxidized RC donor (D+), a bacteriochlorophyll dimer, in the co-crystals in approximately 0.9 micros, which is the same time as measured in solution. This provides strong evidence that the structure of the complex in the region of electron transfer is the same in the crystal and in solution. X-ray diffraction data were collected from co-crystals to a maximum resolution of 2.40 A and refined to an R-factor of 22% (R(free)=26%). The structure shows the cyt c2 to be positioned at the center of the periplasmic surface of the RC, with the heme edge located above the bacteriochlorophyll dimer. The distance between the closest atoms of the two cofactors is 8.4 A. The side-chain of Tyr L162 makes van der Waals contacts with both cofactors along the shortest intermolecular electron transfer pathway. The binding interface can be divided into two domains: (i) A short-range interaction domain that includes Tyr L162, and groups exhibiting non-polar interactions, hydrogen bonding, and a cation-pi interaction. This domain contributes to the strength and specificity of cyt c2 binding. (ii) A long-range, electrostatic interaction domain that contains solvated complementary charges on the RC and cyt c2. This domain, in addition to contributing to the binding, may help steer the unbound proteins toward the right conformation.     

The reaction domain on Rhodospirillum rubrum cytochrome c2 and horse cytochrome c for the Rhodospirillum rubrum cytochrome bc1 complex

The interaction of the Rhodospirillum rubrum cytochrome bc1 complex with R. rubrum cytochrome c2 and horse cytochrome c was studied using specific lysine modification and ionic strength dependence methods. In order to define the reaction domain on cytochrome c2, several fractions consisting of mixtures of singly labeled carboxydintrophenyl-cytochrome c2 derivatives were employed. Fraction A consisted of a mixture of derivatives modified at lysines 58, 81, and 109 on the back of cytochrome c2, while fractions C1, C2, C3, and C4 were mixtures of singly labeled derivatives modified at lysines 9, 13, 75, 86, and 88 on the front of cytochrome c2 surrounding the heme crevice. The rate of the reaction of fraction A was found to be nearly the same as that of native cytochrome c2. However, the rate constants of fractions C1-C4 were found to be more than 20-fold smaller than that of native cytochrome c2. These results indicate that lysine residues surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site on the cytochrome bc1 complex. Since the same domain is involved in the reaction with the photosynthetic reaction center, cytochrome c2 must undergo some type of rotational or translational diffusion during electron transport in R. rubrum. The reaction rates of horse heart cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were also measured. Modification of lysines 8, 13, 25, 27, 72, 79, and 87 surrounding the heme crevice was found to significantly lower the rate of the reaction, while modification of lysines in other regions had no effect. This indicates that the reaction of horse cytochrome c also involves the heme crevice domain.     

The interactions between cytochrome c and cytochrome oxidase that determine the conformation of the oxidized oxidase.

1. Cytochrome c2+ increases the rate at which cytochrome oxidase (EC 1.9.3.1) gamma max428nm) converts to its conformational isomer (gamma max 418-423 nm) but cytochrome c3+ has little effect on the conversion rate. 2. Interactions between reduced cytochrome oxidase and cytochrome c were studied in the absence of electron flow using anaerobic Sephadex columns. 3. Oxidase that is reduced by cytochrome c2+ or other reductant forms the 418-to 423-nm isomer if its last contact, before oxidation, is with cytochrome c3+. If the reduced oxidase contacts cytochrome c2+, before oxidation, the 428-nm oxidase forms.     

Quantitative affinity chromatographic studies of mitochondrial cytochrome c binding to bacterial photosynthetic reaction center, reconstituted in liposome membranes and immobilized by detergent dialysis and avidin--biotin binding

In order to study the affinity binding of c-type cytochromes to the photosynthetic reaction center (RC) by quantitative affinity chromatography (QAC), RC from Rhodobacter sphaeroides was reconstituted into liposomes composed of egg phosphatidylcholine (EPC) and 2 mol% of biotinyl phosphatidylethanolamine simultaneously as the liposomes were formed and immobilized in (strept)avidin-coupled gel beads by rotary detergent dialysis. The immobilized amount was up to 80 nmol of RC and 33 micromol of lipid/g of moist gel in streptavidin-coupled Sephacryl S-1000 gel. By QAC frontal runs, retardation of mitochondrial cyt c on immobilized RC liposome columns was demonstrated. The dissociation constant for the RC-cyt c interaction was determined to be 0.20-0.57 microM. QAC studies also allowed evaluation of the orientation of reconstituted RC in immobilized liposomes by comparison of the total amount of cyt c binding sites with the amount of available binding sites obtained by QAC. It seems that the RC proteoliposomes immobilized in Sephacryl S-1000 gel exposed the cyt c binding sites on the outer surface of the liposomes due to effects of the gel network pore size and the resulting liposomal size.     

Use of specific lysine modifications to identify the site of reaction between cytochrome c and ferricyanide

The site of the reaction between horse heart ferrocytochrome c and ferricyanide was investigated by measuring the reaction rate of cytochrome c derivatives specifically modified at single lysine residues to form trifluoroacetyl or trifluoromethylphenylcarbamyl amino groups. Cytochrome c derivatives singly modified at lysines 8, 13, 25, 27, 72, 79, and 87 surrounding the heme crevice had rate constants decreased from that of native cytochrome c by factors of 1.29, 2.03, 1.12, 1.35, 1.46, 1.29, and 1.19, respectively. Modification of a given lysine with the bulky trifluoromethylphenylcarbamyl group caused nearly the same decrease in reaction rate as modification with the trifluoroacetyl group, indicating that the effect was due to removal of an electrostatic interaction between the protonated lysine amino group and ferricyanide. Modification of lysines 22, 55, 99, and 100 at the right side, bottom, and back of cytochrome c had no effect on the reaction rate. These results indicate that the reaction site is located at the exposed edge of the heme and that the electrostatic interaction between ferricyanide and cytochrome c is dominated by the lysine amino groups surrounding the heme crevice, which include lysine 86, in addition to the ones listed above. We have used the specific lysine modification results to estimate the contribution of each lysine amino group to the electrostatic interaction and have developed a semiempirical relation for the total electrostatic interaction.     

Reconstitution of the heliobacterial photochemical reaction center and cytochrome c553 into a proteoliposome system

Photosynthesis Research - The heliobacterial reaction center (HbRC) is the simplest known photochemical reaction center, in terms of its polypeptide composition. In the heliobacterial cells, its...