Role of cooperative H(+)/e(-) linkage (redox bohr effect) at heme a/Cu(A) and heme a(3)/Cu(B) in the proton pump of cytochrome c oxidase

It is a pleasure to contribute to the special issue published in honor of Vladimir Skulachev, a distinguished scientist who greatly contributes to maintain a high standard of biochemical research in Russia. A more particular reason can be found in his work (Artzabanov, V. Y., Konstantinov, A. A., and Skulachev, V. P. (1978) FEBS Lett., 87, 180–185), where observations anticipating some ideas presented in my article were reported. Cytochrome c oxidase exhibits protonmotive, redox linked allosteric cooperativity. Experimental observations on soluble bovine cytochrome c oxidase are presented showing that oxido-reduction of heme a/Cu_A and heme a ₃/Cu_B is linked to deprotonation/protonation of two clusters of protolytic groups, A₁ and A₂, respectively. This cooperative linkage (redox Bohr effect) results in the translocation of 1 H⁺/oxidase molecule upon oxido-reduction of heme a/Cu_A and heme a ₃/Cu_B, respectively. Results on liposome-reconstituted oxidase show that upon oxidation of heme a/Cu_A and heme a ₃/Cu_B protons from A₁ and A₂ are released in the outer aqueous phase. A₁ but not A₂ appears to take up protons from the inner aqueous space upon reduction of the respective redox center. A cooperative model is presented in which the A₁ and A₂ clusters, operating in close sequence, constitute together the gate of the proton pump in cytochrome c oxidase.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 220–230.Original Russian Text Copyright © 2005 by Papa.This revised version was published online in April 2005 with corrections to the post codes.     

Structure and coordination of CuB in the Acidianus ambivalens aa 3 quinol oxidase heme–copper center

The coordination environment of the Cu_B center of the quinol oxidase from Acidianus ambivalens, a type B heme–copper oxygen reductase, was investigated by Fourier transform (FT) IR and extended X-ray absorption fine structure (EXAFS) spectroscopy. The comparative structural chemistry of dinuclear Fe–Cu sites of the different types of oxygen reductases is of great interest. Fully reduced A. ambivalens quinol oxidase binds CO at the heme a ₃ center, with ν(CO)=1,973 cm⁻¹. On photolysis, the CO migrated to the Cu_B center, forming a Cu_B^I–CO complex with ν(CO)=2,047 cm⁻¹. Raising the temperature of the samples to 25°C did not result in a total loss of signal in the FTIR difference spectrum although the intensity of these signals was reduced sevenfold. This observation is consistent with a large energy barrier against the geminate rebinding of CO to the heme iron from Cu_B, a restricted limited access at the active-site pocket for a second binding, and a kinetically stable Cu_B–CO complex in A. ambivalens aa ₃. The Cu_B center was probed in a number of different states using EXAFS spectroscopy. The oxidized state was best simulated by three histidines and a solvent O scatterer. On reduction, the site became three-coordinate, but in contrast to the bo ₃ enzyme, there was no evidence for heterogeneity of binding of the coordinated histidines. The Cu_B centers in both the oxidized and the reduced enzymes also appeared to contain substoichiometric amounts (0.2 mol equiv) of nonlabile chloride ion. EXAFS data of the reduced carbonylated enzyme showed no difference between dark and photolyzed forms. The spectra could be well fit by 2.5 imidazoles, 0.5 Cl⁻ and 0.5 CO ligands. This arrangement of scatterers would be consistent with about half the sites remaining as unligated Cu(his)₃ and half being converted to Cu(his)₂Cl⁻CO, a 50/50 ratio of Cu(his)₂Cl⁻ and Cu(his)₃CO, or some combination of these formulations. Electronic Supplementary Material Supplementary material is available for this article at .     

Structural characterization of a binuclear center of a Cu-containing NO reductase homologue from Roseobacter denitrificans: EPR and resonance Raman studies

Aerobic phototrophic bacterium Roseobacter denitrificans has a nitric oxide reductase (NOR) homologue with cytochrome c oxidase (CcO) activity. It is composed of two subunits that are homologous with NorC and NorB, and contains heme c, heme b, and copper in a 1:2:1 stoichiometry. This enzyme has virtually no NOR activity. Electron paramagnetic resonance (EPR) spectra of the air-oxidized enzyme showed signals of two low-spin hemes at 15 K. The high-spin heme species having relatively low signal intensity indicated that major part of heme b₃ is EPR-silent due to an antiferromagnetic coupling to an adjacent Cu_B forming a Fe-Cu binuclear center. Resonance Raman (RR) spectrum of the oxidized enzyme suggested that heme b₃ is six-coordinate high-spin species and the other hemes are six-coordinate low-spin species. The RR spectrum of the reduced enzyme showed that all the ferrous hemes are six-coordinate low-spin species. ν(Fe-CO) and ν(C-O) stretching modes were observed at 523 and 1969 cm⁻¹, respectively, for CO-bound enzyme. In spite of the similarity to NOR in the primary structure, the frequency of ν(Fe-CO) mode is close to those of aa₃- and bo₃-type oxidases rather than that of NOR.     

Identification of b,c, and d cytochromes in the membrane of Vitreoscilla

Cytochromes b, c, d, and o were identified by spectroscopic analysis of respiratory membrane fragments from Vitreoscilla sp., strain C1. Carbon monoxide difference spectra of the reduced membranes had absorption maxima at 416, 534, and 571 nm (ascribed to cytochrome o) and 632 nm (cytochrome d). Derivative spectra of the pyridine hemochromogen spectra of the membranes identified the presence of b- and c-type cytochromes in Vitreoscilla. The cyanide binding curve of the membranes was biphasic with dissociation constants of 2.14 mM and 10.7 mM which were assigned to cytochrome o and cytochrome d, respectively. Membranes bound carbon monoxide with dissociation constant 3.9 M, which was assigned to cytochrome o. Cytochrome c ₅₅₆ and a NADH-p-iodonitrotetrazolium violet reductase component were partially purified from Vitreoscilla membranes.Abbreviations INT p-iodonitrotetrazolium violet - RMF respiratory membrane fragments - K _d dissociation constant - CHAPS 3-[(3-cholamido propyl) dimethylammonio]-1-propanesulfonate - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Cytochrome aa 3 from Methylococcus capsulatus (Bath)

A cytochrome aa ₃-type oxidase was isolated with and without a c-type cytochrome (cytochrome c-557) from Methylococcus capsulatus Bath by ion-exchange and hydrophobic chromatography in the presence of Triton X-100. Although cytochrome c-557 was not a constitutive component of the terminal oxidase, the cytochrome c ascorbate-TMPD oxidase activity of the enzyme decreased dramatically when the ratio of cytochrome c-557 to heme a dropped below 1:3. On denaturing gels, the purified enzyme dissociated into three subunits with molecular weights of 46,000, 28,000 and 20,000. The enzyme contains two heme groups (a and a ₃), absorption maximum at 422 nm in the resting state, at 445 and 601 nm in the dithionite reduced form and at 434 and 598 nm in the dithionite reduced plus CO form. Denaturing gels of the cytochrome aa ₃-cytochrome c-557 complex showed the polypeptides associated with cytochrome aa ₃ plus a heme c-staining subunit with a molecular weight of 37,000. The complex contains approximately two heme a, one heme c, absorption maximum at 420 nm in the resting state and at 421, 445, 522, 557 and 601 nm in the dithionite reduced form. The specific activity of the purified enzyme was 130 mol O₂/min · mol heme a compared to 753 mol O₂/min · mol heme a when isolated with cytochrome c-557.Abbreviations MMO methan monooxygenase - sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - TMPD N,N,N,N-tetramethyl-p-phenylenediamine dihydrochloride - Na₂EDTA disodium ethylenediamine-tetraacetic acid     

How rapid are the internal reactions of the ubiquinol:cytochrome c 2 oxidoreductase?

The temperature dependence of the partial reactions leading to turn-over of the UQH₂:cyt c ₂ oxidoreductase of Rhodobacter sphaeroides have been studied. The redox properties of the cytochrome components show a weak temperature dependence over the range 280–330 K, with coefficients of about 1 m V per degree; our results suggest that the other components show similar dependencies, so that no significant change in the gradient of standard free-energy between components occurs over this temperature range. The rates of the reactions of the high potential chain (the Rieske iron sulfur center, cytochromes c ₁ and c ₂, reaction center primary donor) show a weak temperature dependence, indicating an activation energy < 8 kJ per mole for electron transfer in this chain. The oxidation of ubiquinol at the Q_z-site of the complex showed a strong temperature dependence, with an activation energy of about 32 kJ mole^–1. The electron transfer from cytochrome b-566 to cytochrome b-561 was not rate determining at any temperature, and did not contribute to the energy barrier. The activation energy of 32 kJ mole^–1 for quinol oxidation was the same for all states of the quinone pool (fully oxidized, partially reduced, or fully reduced before the flash). We suggest that the activation barrier is in the reaction by which ubiquinol at the catalytic site is oxidized to semiquinone. The most economical scheme for this reaction would have the semiquinone intermediate at the energy level indicated by the activation barrier. We discuss the plausibility of this simple model, and the values for rate constants, stability constant, the redox potentials of the intermediate couples, and the binding constant for the semiquinone, which are pertinent to the mechanism of the ubiquinol oxidizing site.Abbreviations (BChl)₂ P870, primary donor of the photochemical reaction center - b/c ₁ complex ubiquinol: cytochrome c ₂ oxidoreductase - cyt b _H cytochrome b-561 or higher potential cytochrome b - cyt b _L cytochrome b-566, or low potential cytochrome b - cyt c ₁, cyt c ₂, cyt c _t cytochromes c ₁ and c ₂, and total cytochrome c (cyt c ₁ and cyt c ₂) - Fe.S Rieske-type iron sulfur center, Q - QH₂ ubiquinone, ubiquinol - Q_z, Q_zH₂, Q_z ^– ubiquinone, ubiquinol, and semiquinone anion of ubiquinone, bound at quinol oxidizing site - Q_z-site ubiquinol oxidizing site (also called Q_o-(outside) - Q_o (Oxidizing) - Q_P (Positive proton potential) site) - Q_c-site uubiquinone reductase site (also called the Q_i-(inside) - Q_R (Reducing), or - Q_N (Negative proton potential) site) - UHDBT 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazol     

Genes for a second terminal oxidase in Bradyrhizobium japonicum

Bradyrhizobium japonicum possesses a mitochondria-like respiratory chain terminating with an aa ₃-type cytochrome c oxidase. The gene for subunit I of this enzyme (coxA) had been identified and cloned previously via heterologous hybridization using a Paracoccus denitrificans DNA probe. In the course of these studies, another B. japonicum DNA region was discovered which apparently encoded a second terminal oxidase that was different from cytochrome aa ₃ but also belonged to the superfamily of heme/copper oxidases. Nucleotide sequence analysis revealed a cluster of at least four genes, coxMNOP, organized most probably in an operon. The predicted coxM gene product shared significant similarity with subunit II of cytochrome c oxidases from other organisms: in particular, all of the proposed Cu_A ligands were conserved as well as three of the four acidic amino acid residues that might be involved in the binding of cytochrome c. The coxN gene encoded a polypeptide with about 40% sequence identity with subunit I representatives including the previously found CoxA protein: the six presumed histidine ligands of the prosthetic groups (two hemes and Cu_B) were strictly conserved. A remarkable feature of the DNA seqence was the presence of two genes, coxO and coxP, whose products were both homologous to subunit III proteins. A B.japonicum coxN mutant strain was created by marker exchange mutagenesis which, however, exhibited no obvious defects in free-living, aerobic growth or in root nodule symbiosis with soybean. This shows that the coxMNOP genes are not essential for respiration in the N₂ fixing bacteroid.Abbreviations ORF open reading frame - TMPD N,N,N',N'-tetramethyl-p-phenylenediamine     

The sequence of electron carriers in the reaction of cytochromec oxidase with oxygen

Kinetic studies of the electron transfer processes performed by cytochrome oxidase have assigned rates of electron transfer between the metal centers involved in the oxidation of ferrocytochromec by molecular oxygen. Transient-state studies of the reaction with oxygen have led to the proposal of a sequence of carriers from cytochromec, to Cu_A, to cytochromea, and then to the binuclear (i.e., cytochromea ₃-Cu_B) center. Electron exchange rates between these centers agree with relative center-to-center distances as follows; cytochromec to Cu_A 5–7 Å, cytochromec to cytochromea 20–25 Å, Cu_A to cytochromea 14–16 Å and cytochromea to cytochrome a₃-Cu_B 8–10 Å. It is proposed that the step from cytochromea to the binuclear center is the key control point in the reaction and that this step is one of the major points of energy transduction in the reaction cycle.     

The Role of Electrostatic Interactions for Cytochrome c Oxidase Function

In recent years, the enormous increase in high-resolution three-dimensional structures of proteins together with the development of powerful theoretical techniques have provided the basis for a more detailed examination of the role of electrostatics in determining the midpoint potentials of redox-active metal centers and in influencing the protonation behavior of titratable groups in proteins. Based on the coordinates of the Paracoccus denitrificans cytochrome c oxidase, we have determined the electrostatic potential in and around the protein, calculated the titration curves for all ionizable residues in the protein, and analyzed the response of the protein environment to redox changes at the metal centers. The results of this study provide insight into how charged groups can be stabilized within a low-dielectric environment and how the range of their electrostatic effects can be modulated by the protein. A cluster of 18 titratable groups around the heme a ₃–Cu_B binuclear center, including a hydroxide ion bound to the copper, was identified that accounts for most of the proton uptake associated with redox changes at the binuclear site. Predicted changes in net protonation were in reasonable agreement with experimentally determined values. The relevance of these findings in the light of possible mechanisms of redox-coupled proton movement is discussed.     

Insight into the active-site structure and function of cytochrome oxidase by analysis of site-directed mutants of bacterial cytochromeaa 3 and cytochromebo

Cytochromeaa ₃ ofRhodobacter sphaeroides and cytochromebo ofE. coli are useful models of the more complex cytochromec oxidase of eukaryotes, as demonstrated by the genetic, spectroscopic, and functional studies reviewed here. A summary of site-directed mutants of conserved residues in these two enzymes is presented and discussed in terms of a current model of the structure of the metal centers and evidence for regions of the protein likely to be involved in proton transfer. The model of ligation of the hemea ₃ (oro)-Cu_B center, in which both hemes are bound to helix X of subunit I, has important implications for the pathways and control of electron transfer.     

Electron donation from membrane-bound cytochrome c to the photosynthetic reaction center in whole cells and isolated membranes of Heliobacterium gestii

The reaction between membrane-bound cytochrome c and the reaction center bacteriochlorophyll g dimer P798 was studied in the whole cells and isolated membranes of Heliobacterium gestii. In the whole cells, the flash-oxidized P798⁺ was rereduced in multiple exponential phases with half times (t _1/2s) of 10 s, 300 s and 4 ms in relative amplitudes of 40, 35 and 25%, respectively. The faster two phases were in parallel with the oxidation of cytochrome c. In isolated membranes, a significantly slow oxidation of the membrane-bound cytochrome c was detected with t _1/2 = 3 ms. This slow rate, however, again became faster with the addition of Mg²⁺. The rate showed a high temperature dependency giving apparent activation energies of 88.2 and 58.9 kJ/mol in the whole cells and isolated membranes, respectively. Therefore, membrane-bound cytochrome c donates electrons to the P798⁺ in a collisional reaction mode like the reaction of water-soluble proteins. The rereduction of the oxidized cytochrome c was suppressed by the addition of stigmatellin both in the whole cells and isolated membranes. This indicates that the electron transfer from the cytochrome bc complex to the photooxidized P798⁺ is mediated by the membrane-bound cytochrome c. The multiple flash excitation study showed that 2–3 hemes c were connected to the P798. By the heme staining after the SDS-PAGE analysis of the membraneous proteins, two cytochromes c were detected on the gel indicating apparent molecular masses of 17 and 30 kDa, respectively. The situation resembles the case in green sulfur bacteria, that is, the membrane-bound cyotochrome c _z couples electron transfer between the cytochrome bc complex and the P840 reaction center complex.This revised version was published online in October 2005 with corrections to the Cover Date.     

Mössbauer spectra of the heme peptide (HP) 1–50 and the heme peptide:non-heme peptide (NHP) non-covalent complex 1–50:51–104 derived from cytochrome c: evidence for cytochrome c iron site solvation in aqueous solution

Mössbauer spectroscopic studies on a heme peptide (HP) derived from cytochrome c and on the HP recombined non-covalently with the remaining cleaved section are reported. The results suggest that the environment of the heme site in the known crystal structure of cytochrome c may differ in detail from the environment of the heme in the working protein.     

Crystal Structure of Bovine Heart Cytochrome c Oxidase at 2.8 Å Resolution

Thirteen different polypeptide subunits, each in one copy, five phosphatidyl ethanolamines and three phosphatidyl glycerols, two hemes A, three Cu ions, one Mg ion, and one Zn ion are detectable in the crystal structure of bovine heart cytochrome c oxidase in the fully oxidized form at 2.8 Å resolution. A propionate of hems a, a peptide unit (–CO–NH–), and an imidazole bound to Cu_A are hydrogen-bonded sequentially, giving a facile electron transfer path from Cu_A to heme a. The O₂ binding and reduction site, heme a ₃, is 4.7 Å apart from Cu_B. Two possible proton transfer paths from the matrix side to the cytosolic side are located in subunit I, including hydrogen bonds and internal cavities likely to contain randomly oriented water molecules. Neither path includes the O₂ reduction site. The O₂ reduction site has a proton transfer path from the matrix side possibly for protons for producing water. The coordination geometry of Cu_B and the location of Tyr²⁴⁴ in subunit I at the end of the scalar proton path suggests a hydroperoxo species as the two electron reduced intermediate in the O₂ reduction process.     

Evolutionary alkaline transition in human cytochrome <Emphasis Type="Italic">c</Emphasis>

Conformational transitions in cytochrome c (cyt c) are being realized to be responsible for its multi-functions. Among a number of conformational transitions in cyt c, the alkaline transition has attracted much attention. The cDNA of human cyt c is cloned by RT-PCR and a high-effective expression system for human cyt c has been developed in this study. The equilibrium and kinetics of the alkaline transition of human cyt c have been systematically investigated for the first time, and compared with those of yeast and horse cyt c from an evolutionary perspective. The pK_a value for the alkaline transition of human cyt c is apparently higher than that of yeast and horse. Kinetic studies suggest that it is increasingly difficult for the alkaline transition of cyt c from yeast, horse and human. Molecular modeling of human cyt c shows that the omega loop where the lysine residue is located apparently further away from heme in human cyt c than in yeast iso-1 and horse heart cyt c. These results regarding alkaline conformational transition provide valuable information for understanding the molecular basis for the biological multi-functions of cyt c.     

Operation of the cbb3-type terminal oxidase in Azotobacter vinelandii

A part of the gene encoding cbb ₃-type cytochrome oxidase CcoN subunit was cloned from Azotobacter vinelandii and a mutant strain of this bacterium with disrupted ccoN gene was constructed. In contrast to the wild type strain, this one is unable to oxidize cytochromes c ₄ and c ₅. Thus, the A. vinelandii respiratory chain is shown to contain cbb ₃-type cytochrome c oxidase. It is also shown that the activity of this enzyme is not necessary for diazotrophic growth of A. vinelandii at high oxygen concentrations.     

Purification of cytochrome a 1 c 1 from Nitrobacter agilis and characterization of nitrite oxidation system of the bacterium

Cytochrome a ₁ c ₁ was highly purified from Nitrobacter agilis. The cytochrome contained heme a and heme c of equimolar amount, and its reduced form showed absorption peaks at 587, 550, 521, 434 and 416 nm. Molecular weight per heme a of the cytochrome was estimated to be approx. 100,000–130,000 from the amino acid composition. A similar value was obtained by determining the protein content per heme a. The cytochrome molecule was composed of three subunits with molecular weights of 55,000, 29,000 and 19,000, respectively. The 29 kd subunit had heme c.Hemes a and c of cytochrome a ₁ c ₁ were reduced on addition of nitrite, and the reduced cytochrome was hardly autoxidizable. Exogenously added horse heart cytochrome c was reduced by nitrite in the presence of cytochrome a ₁ c ₁; K _m values of cytochrome a ₁ c ₁ for nitrite and N. agilis cytochrome c were 0.5 mM and and 6 M, respectively. V _max was 1.7 mol ferricytochrome c reduced/min·mol of cytochrome a ₁ c ₁ The pH optimum of the reaction was about 8. The nitrite-cytochrome c reduction catalyzed by cytochrome a ₁ c ₁ was 61% and 88% inhibited by 44M azide and cyanide, respectively. In the presence of 4.4 mM nitrate, the reaction was 89% inhibited. The nitrite-cytochrome c reduction catalysed by cytochrome a ₁ c ₁ was 2.5-fold stimulated by 4.5 mM manganous chloride. An activating factor which was present in the crude enzyme preparation stimulated the reaction by 2.8-fold, and presence of both the factor and manganous ion activated the reaction by 7-fold.Cytochrome a ₁ c ₁ showed also cytochrome c-nitrate reductase activity. The pH optimum of the reaction was about 6. The nitrate reductase activity was also stimulated by manganous ions and the activating factor.     

Pathways of Proton Transfer in Cytochrome c Oxidase

During the last few years our knowledge of the structure and function of heme copper oxidases has greatly profited from the use of site-directed mutagenesis in combination with biophysical techniques. This, together with the recently-determined crystal structures of cytochrome c oxidase, has now made it possible to design experiments aimed at targeting specific pump mechanisms. Here, we summarize results from our recent kinetic studies of electron and proton-transfer reactions in wild-type and mutant forms of cytochrome c oxidase from Rhodobacter sphaeroides. These studies have made it possible to identify amino acid residues involved in proton transfer during specific reaction steps and provide a basis for discussion of mechanisms of electron and proton transfer in terminal oxidases. The results indicate that the pathway through K(I-362)/T(I-359), but not through D(I-132)/E(I-286), is used for proton transfer to a protonatable group interacting electrostatically with heme a ₃, i.e., upon reduction of the binuclear center. The pathway through D(I-132)/E(I-286) is used for uptake of pumped and substrate protons during the pumping steps during O₂ reduction.     

Reactivity of Nitric Oxide with Cytochrome c Oxidase: Interactions with the Binuclear Centre and Mechanism of Inhibition

Nitric oxide (NO) has recently been recognized as an important biological mediator that inhibits respiration at cytochrome c oxidase (CcO). This inhibition is reversible and shows competition with oxygen, the K _i being lower at low oxygen concentrations. Although the species that binds NO in turnover has been suggested to contain a partially reduced binuclear center, the exact mechanism of the inhibition is not clear. Recently, rapid (ms) redox reactions of NO with the binuclear center have been reported, e.g., the ejection of an electron to cytochrome a and the depletion of the intermediates P and F. These observations have been rationalized within a scheme in which NO reacts with oxidized Cu_B leading to the reduction of this metal center and formation of nitrite in a very fast reaction. Electron migration from Cu_B to other redox sites within the enzyme is proposed to explain the optical transitions observed. The relevance of these reactions to the inhibition of CcO and metabolism of NO are discussed.     

Respiratory deficient mutants of Rhodopseudomonas capsulata

The facultative photosynthetic bacterium Rhodopseudomonas capsulata was mutagenized by transfer of the plasmid pSUP201::Tn5 from Escherichia coli to R. capsulata. Mutants defective in cytochrome oxidase and other respiratory functions were selected by replica plating, NADI-reaction and immunological methods. Among 20,000 mutants no clone was detected, which lacks the 65,000-protein of the cytochrome oxidase, but many mutants have been isolated which were cytochrome oxidase deficient (or inactive). Other mutants excrete heme and cytochrome c into the medium and lack cytochrome c ₂.Abbreviations Ap ampicillin - CIE crossed immunoelectrophoresis - cyt cytochrome - Cm chloramphenicol - Km kanamycin - SDS sodium dodecylsulfate - Tc tetracycline     

Interaction of horse cytochromec with the photosynthetic reaction center ofRhodospirillum rubrum

Mitochondrial cytochromec (horse), which is a very efficient electron donor to bacterial photosynthetic reaction centersin vitro, binds to the reaction center ofRhodospirillum rubrum with an approximate dissociation constant of 0.3–0.5 µM at pH 8.2 and low ionic strength. The binding site for the reaction center is on the frontside of cytochromec which is the side with the exposed heme edge, as revealed by differential chemical acetylation of lysines of free and reaction-center-bound cytochromec. In contrast, bacterial cytochromec ₂ was found previously to bind to the detergent-solubilized reaction center through its backside, i.e., the side opposite to the heme cleft [Rieder, R., Wiemken, V., Bachofen, R., and Bosshard, H. R. (1985).Biochem. Biophys. Res. Commun. 128, 120–126]. Binding of mitochondrial cytochromec but not of mitochondrial cytochromec ₂ is strongly inhibited by low concentrations of poly-l-lysine. The results are difficult to reconcile with the existence of an electron transfer site on the backside of cytochromec ₂.