A map of dielectric heterogeneity in a membrane protein: the hetero-oligomeric cytochrome b6f complex

Abstract

The cytochrome b6f complex, a member of the cytochrome bc family that mediates energy transduction in photosynthetic and respiratory membranes, is a hetero-oligomeric complex that utilizes two pairs of b-hemes in a symmetric dimer to accomplish trans-membrane electron transfer, quinone oxidation-reduction, and generation of a proton electrochemical potential. Analysis of electron storage in this pathway, utilizing simultaneous measurement of heme reduction, and of circular dichroism (CD) spectra, to assay heme-heme interactions, implies a heterogeneous distribution of the dielectric constants that mediate electrostatic interactions between the four hemes in the complex. Crystallographic information was used to determine the identity of the interacting hemes. The Soret band CD signal is dominated by excitonic interaction between the intramonomer b-hemes, bn and bp, on the electrochemically negative and positive sides of the complex. Kinetic data imply that the most probable pathway for transfer of the two electrons needed for quinone oxidation-reduction utilizes this intramonomer heme pair, contradicting the expectation based on heme redox potentials and thermodynamics, that the two higher potential hemes bn on different monomers would be preferentially reduced. Energetically preferred intramonomer electron storage of electrons on the intramonomer b-hemes is found to require heterogeneity of interheme dielectric constants. Relative to the medium separating the two higher potential hemes bn, a relatively large dielectric constant must exist between the intramonomer b-hemes, allowing a smaller electrostatic repulsion between the reduced hemes. Heterogeneity of dielectric constants is an additional structure-function parameter of membrane protein complexes.

Figure 1

(A) Cytochrome b₆f complex structure from Nostoc PCC 7120 (PDB ID 4H44). Ribbon diagram of polypeptide subunits and redox active groups. Cytochrome b₆ subunit, yellow; subunitIV, orange; cytochrome f, red; Rieske [2Fe–2S] iron–sulfur protein, blue; PetG, pink; PetL, wheat; PetM, cyan; PetN, white. Hemes b_p and b_n (green, red, and blue), c_n (black, red, and blue), f (gray, red, and blue), chlorophyll-a (dark green and blue), and β-carotene (yellow) are shown as sticks. The components of the [2Fe–2S] cluster of the Rieske iron–sulfur protein are depicted as spheres (Fe, brown; sulfur, yellow). (B) Geometry of hemes within the trans-membrane domain of cytochrome b₆f (PDB 4H44). Edge–edge and center–center (Fe–Fe, in parentheses) distances are shown.

Figure 2

Absorbance spectra of dithionite-reduced cytochrome b₆f complex; simultaneous kinetics of reduction of hemes b_n and b_p in dimeric b₆f complex. (A) Room temperature α-band absorbance spectra of cytochromes f and b₆, with room temperature absorbance spectra, respectively, at 554 and 563 nm, as a function of time of dimeric b₆f complex initially oxidized by ferricyanide (FeCy; 20 μM), reduced by ascorbate (Asc; 0.1 mM), and reduced subsequently by dithionite (Dth, final concentration 2–3 mM). Buffer, 30 mM HEPES, pH 7.5, 50 mM NaCl, 0.1 mM EDTA, 0.045% UDM. Dimeric b₆f complex containing 0.8 μM cytochrome f. (B, C) Time course of (B) heme b reduction by dithionite and (C) increase in amplitude of the split CD spectrum in dimeric b₆f complex; kinetics of heme b reduction by dithionite. The dimeric b₆f complex contained 1.6 μM cytochrome f. Dithionite was added to a concentration of 3–4 mM, and 27 consecutive OD and CD spectra (420–440 nm) were simultaneously measured (16 s per spectrum). Measurement of all CD and OD difference spectra was started 10 (blue), 74 (pink), 138 (green), 234 (red), and 410 (brown) s after addition of dithionite. (D) Measured kinetics induced by dithionite addition to the dimeric b₆f complex: absorbance increase at 432 nm (triangles, black); amplitude of Soret band circular dichroism (CD) signals at 428 nm (circles, blue) and 437 nm (squares, blue).

Figure 3

Calculated CD interaction between all reduced heme pairs: p1–n1 or p2–n2 (red trace), which are the dominant interactions; calculated spectra are shown for the other heme pairs, p1–n2, p2–n1 (purple), p1–p2 (blue), and n1–n2 (green).

Figure 4

CD (circles, squares, blue) and optical density changes (triangles, black) were measured simultaneously, as described in the Materials and Methods section. Solid curves are kinetic simulations as described in the Results section, with the assumption that dithionite acts as a one-electron donor; the predicted time course for dithionite acting as a two-electron donor is shown in Figure S7 (Supporting Information). Electron transfer to the b₆f dimer is described by two rate constants: k₁ = 0.017 s^–1 (70%) and k₂ = 0.33 s^–1 (30%); the larger rate constant is attributed to “contaminant” monomer complex in the dimer preparation (Figure S2B, Supporting Information, native gel). Black curve: fit to measured absorbance changes (triangles). Blue curve: expected CD kinetics in the “n–p” model where the intermediate doubly reduced intramonomer (Figure 5B; state N₂) dimer has a lower free energy than the intermonomer doubly reduced state in which heme b_n is reduced in both monomers (Figure 5A; state N₂). Red function: expected time course of the CD change in the “n–n” model in which the intramonomer n1–n2 doubly reduced state has the lowest free energy.

Figure 5

Summary of possible electron transfer routes and heme reduction states in the b₆f complex. Two different models are considered: (A) n1–n2 model: the doubly reduced state of lowest energy of the dimer corresponds to two electrons residing on the two b_n hemes belonging to different subunits (this state produces a weak negative CD signal (Figure 3). (B) n–p model: the lowest doubly reduced state of the dimer corresponds to two electrons residing on the b_n and b_p hemes belonging to the same subunit (in this state, the amplitude of the positive CD signal is significantly larger than that of any other heme pair). The sequence of four electron transfer events in these two models is illustrated. Reduced hemes are shown as red spheres. State N₀ in panels A and B denotes fully oxidized hemes in dimeric complex. N_i represent states of the dimeric complex in which subscript “i” represents the number of reduced hemes. States N₁ and N₃ are bypassed if dithionite acts as a 2 e^– donor. (C) Summary of conceivable two electron half-reduced states, of which the three states marked by “X” are inferred to be substantially less probable, although they have been documented to exist (refs (14), (15), (17), (19), (21), and (26)).

Figure 6

Description of dielectric heterogeneity in the cytochrome b₆f complex (PDB ID 4H44). Four interheme dielectric constants, which have different values, are shown: (i) the reference dielectric constant, ε_n1,_n2 ≡ 2.5 between the two n-side hemes that bridge the major intermonomer cavity (yellow) that contains a high concentration of lipid (Hasan et al., submitted for publication), (ii) ε_n1,_p1 = ε_n2,_p2 between the intramonomer hemes, (iii) ε_p1,n2 = ε_p2,n1 between the p-side heme on one monomer and the n-side heme on the other, and (iv) ε_p1,_p2 between the two p-side hemes. The minimum values of ε necessary for the energetically favored reduction of a particular heme pair among the four possible pairs in the dimeric complex, dependent upon the midpoint redox potential difference (ΔE_m) between hemes b_n and b_p (50, 75, and 100 mV), calculated to the nearest half-integral values using the reference dielectric constant for ε_n1,n2 of 2.5 and eq 9 are, respectively, ε_n1,p1 = ε_n2,p2 > 6.1, 7.8, and 10.8 (Table 2). The corresponding values for the electrostatic interaction between the two hemes on different monomers on opposite sides of the complex, ε_n1,p2 = ε_n2,p1, are >3.7, 4.7, and 6.6. Energetically preferred reduction of the p-side heme pair, p1 and p2, corresponding to a ΔE_m between hemes b_n and b_p of 50 and 75 mV, respectively, would require dielectric constants >10.1 and >43.6.