Thylakoid Membrane Protein Topography LOCATION OF THE TERMINI OF THE CHLOROPLAST CYTOCHROME bs ON THE STROMAL SIDE OF THE MEMBRANE*

The orientation of cytochrome bs in the thylakoid membrane and the question of whether the number of membrane spanning helices is an even or odd number was tested through the relative trypsin susceptibility of epitopes (Asp-5 to Gln-14) and (Ile-205 to Leu-214) at the NH2 and COOH termini, respectively, of the 214-residue cytochrome be polypeptide. A structure of the cytochrome with an even number of helices and the NH, and COOH termini on the stromal side of the membrane was inferred from the following: The sensitive

The orientation of cytochrome bs in the thylakoid membrane and the question of whether the number of membrane spanning helices is an even or odd number was tested through the relative trypsin susceptibility of epitopes  and  to Leu-214) at the NH2 and COOH termini, respectively, of the 214residue cytochrome be polypeptide. A structure of the cytochrome with an even number of helices and the NH, and COOH termini on the stromal side of the membrane was inferred from the following: 1) cleavage of cytochrome be by trypsin added to thylakoids occurs by removal of both of the exposed NH2-and COOH-terminal epitopes.
The epitopes at the termini were more sensitive to trypsin after prior treatment of thylakoids with carboxypeptidase A, indicating that these epitopes are shielded on the stromal side of the membrane by the COOH termini of other proteins.
2) Both epitopes were more trypsin-sensitive in thylakoid membranes than was cytochrome f that is only sensitive to trypsin acting on the lumen side of the membrane.
3) The NH2-and COOH-terminal epitopes of cytochrome be were also more sensitive to trypsin added to thylakoid membranes than were the oxygenevolving complex 16-and 33-kDa proteins that are completely located on the lumen side. 4) The order of trypsin susceptibility was reversed in inside-out membranes, where the cytochrome NH2-and COOH-terminal epitopes were less sensitive than the 16-and 33-kDa proteins.
The decreased relative sensitivity of the cytochrome be epitopes occurs in spite of a greater absolute sensitivity of these epitopes to trypsin in inside-out membranes. 5) The greater absolute sensitivity can be explained by a 4-helix model that includes trypsin-sensitive sites on the lumen side.
The multisubunit mitochondrial cytochrome bcI complex and the b6f complex of oxygenic photosynthesis occupy a central position in the respective electron transport chains, between protein complexes supplying low potential reducing equivalents (dehydrogenases and photosystem II, respectively) and those acting as the terminal oxidizing complex * This work was supported by National Institutes of Health Grant GM-38323 and a grant to the Structural Biology Group of this department from the Lucille P. Markey Charitable Trust. (cytochrome oxidase and the photosystem I reaction center, respectively) (Hauska et al., 1983;Cramer et al., 1987). The bcl complex of photosynthetic bacteria functions in a cyclic pathway in which the reaction center supplies both reductant and oxidant on the n-and p-sides' of the truns-membrane complex (Crofts, 1985). The primary sequences of cytochrome b of the mitochondrial and bacterial bcl complex (consisting of 379-434 residues except for trypanosomes (Hauska et al., 1988)), that will be referred to subsequently as "cytochrome b(bcl)," have a high degree of identity. Cytochrome bs is approximately half the size of cytochrome b(bcJ. The sequences of the chloroplast-cyanobacterial cytochromes bs are similar, but show a smaller degree of identity when compared to the sequences of cytochrome b(bcJ. The smaller 214-residue chloroplast cytochrome bs provided the key to identifying the 4 histidine ligands needed for heme coordination (Widger et al., 1984), since it only contains 4 His residues in hydrophobic domains and a total of only 5 in the spinach chloroplast sequence originally analyzed (Alt and Herrmann, 1984). The distribution of hydrophobic segments in the hemebinding domain was found to be highly correlated between cytochrome bs and cytochrome b(bcI) (Widger et al., 1984), leading to the inferences (a) that the 4 His residues used in heme coordination are located on two of the 19-23-residue membrane-spanning cu-helices, with a His pair coordinated to a heme on each side of the membrane that bridge these two helices (Widger et al., 1984;Saraste, 1984);and (b) five and nine helices are utilized, respectively, for the folding of the cytochrome b6 (Fig. lA) and cytochrome b(bcl) polypeptides in the membrane bilayer, with the histidines coordinating the hemes residing on helices II and V. In these models, the amino acid composition of helix IV of cytochrome b6 is unusually polar (1 Asp, 1 Glu, 1 Ser, 1 Thr, 2 Gly, and 3 Pro in the spinach chloroplasts).
Subsequent analysis of the location in the cytochrome b(bcJ polypeptide of inhibitor-resistant mutations in yeast (Di Rago and Colson, 1988;Di Rago et al., 1989) and mouse (Howell and Gilbert, 1988) mitochondria, and in the photosynthetic bacterium Rhodobucter cupsulutus (Daldal, 1987;Daldal et al., 1989), indicated (a) that the cytochromes are oriented with the NH2 terminus of these proteins on the n-side of the membrane, and (b) that these cytochromes contain eight membrane-spanning cY-helices instead of the nine originally proposed. This analysis was based on the use of inhibitors of the cytochrome bcl complex that are thought to specifically compete for quinone binding sites on the n-or p-side of the membrane.
The data on inhibitor-resistant mutants would It is assumed in this drawing that the NH, terminus is on the n-(stromal) side of the membrane, although this was proven only for cytochrome b, in the present work.
imply by analogy that the cytochrome bs polypeptide contains four trans-membrane helices ( Fig. IA) instead of five in the original model (Fig. 1B). One problem with the interpretation of the mutant data is that the action of n-andp-side inhibitors is not well defined because mutant loci are found that map in the center of the bilayer (cf. Fig. 4 in Daldal et al., 1989).
The genetic data for the bacterial and mitochondrial b cytochromes have not yet been checked by biochemical methods of topographical analysis. Furthermore, it is important to independently determine the topography of the cytochrome b6 polypeptide because relative to cytochrome b(bcJ there are significant structural  and functional differences. Regarding the latter, cytochrome bs unlike cytochrome b(bcJ does not bind quinone photoaffinity probes (Doyle et at., 1989), and there is evidence that electron transfer between the hemes is not observed (Furbacher et al., 1989). The use of inhibitor-resistant mutants in cyanobacteria to check the topography of the cytochrome bs polypeptide will be difficult because of the absence or paucity of n-side inhibitors (Furbacher et at., 1989). The present work uses the accessibility/sensitivity to trypsin of epitopes on the cytochrome bs polypeptide as probes of its orientation in the membrane.
A preliminary and abbreviated version of this work has been presented (Szczepaniak and Cramer, 1990).

MATERIALS AND METHODS
Preparation of Thylakoids, Inside-out, Right Side-out Membranes Preparation of Thylakoids-Spinach leaves grown on a 12-h lightdark cycle (50 g) were homogenized in 250 ml of buffer (0.3 M sucrose, 10 mM NaCl, 50 mM Hepes, pH 7.5) for about 5 s, filtered through four layers of cheesecloth, then subjected to centrifugation at 1000 X g (3 min, and immediately stopped by hand). The sediment was resuspended in 50 mM sucrose, 10 mM NaCl, and 50 mM Hepes, pH 7.5, centrifuged again at 1000 x g for 30 s, the sediment discarded, and the supernatant centrifuged (3000 X g, 5 min). The resultant centrifuge pellet was resuspended in homogenization buffer in which it was incubated for at least 30 min to ensure a high degree of unstacking prior to treatment with trypsin or carboxypeptidase. Assay of Chloroplast Stacking-The relative degree of chloroplast thylakoid stacking was monitored by the ability of digitonin to disrupt the membrane systems (Chow and Barber, 1980;Chow et al., 1980). Chloroplasts (0.4 mg/ml) were resuspended in homogenization buffer, allowed to stand for 30 min in 0 "C, and then incubated with 0.5% digitonin at 0 "C for 30 min. The mixture was then diluted 6-fold with the same buffer and centrifuged at 10,000 X g for 30 min. 5 mM), and then centrifuged at 1,000 X g (10 min) to remove starch.
After addition of thylakoid homogenate (4 mg of chlorophyll in 1 ml added to 24 ml), the phase system was mixed and centrifuged at 1,500 X g (3 min). The upper (Tl) and lower (Bl) phase were collected and repartitioned with pure lower or upper phase, respectively, two more times to generate fractions T3 and B3 (notation of Andersson and Akerlund, 1978) corresponding to right side-out and inside-out vesicles. Vesicles of each type were diluted with phase partition buffer and sedimented at 100,000 x g (30 min).

Peptide Synthesis
Two decapeptides, corresponding to epitopes spanning residues 5-14 and 205-214, respectively, of the 214-amino acid cytochrome bg polypeptide from spinach chloroplasts were synthesized and coupled to bovine serum albumin or keyhole limpet hemocyanin as previously described for the generation of antibodies in rabbits or chickens (Szczepaniak et al., 1989). The position of these epitopes in a 5-or 4-helix model for cytochrome b6 folding in the membrane is shown (Fig. 1). In addition, antibody III to the entire cytochrome b, polypeptide was prepared to cytochrome b6 electroeluted from an SDS gel of purified

SDS-PAGE and Western Blotting
The control and protease-treated thylakoid membranes were extracted with 90% acetone, recovered by centrifugation (4 min, 10,000 X g) at 4 "C, and were solubilized prior to electrophoresis in buffer containing 4 M urea, 2% SDS, 10% glycerol, 5% P-mercaptoethanol, and 50 mM Tris, pH 8.6, at a final chlorophyll concentration of 1 mg/ ml. Proteins were separated electrophoretically in a gel system using 15% acrylamide (Piccioni et al., 1982). The gel was incubated in 0.025 M Tris, 0.193 M glycine, and 20% methanol, and the Western transfer was done at 100 mA for 2 h, or 135 mA for 30 min using a semidry transfer unit (Hoefer). The nitrocellulose paper (Amersham Hybond) was then soaked in Tris-buffered saline buffer (10 mM Tris, pH 7.5, 0.15 M NaCl) with 0.25% gelatin and 5% powdered nonfat dry milk for at least 3 h (usually overnight). After equilibration with antibody in Tris-buffered saline and 0.25% gelatin for 2 h, and washing in Tris-buffered saline containing 0.05% Nonidet P-40, the paper was reacted (2 h) with a second antibody conjugated with peroxidase in 0.25% gelatin and the Tris saline, washed in Tris saline containing 0.05% Nonidet P-40, and stained (10 min) with 0.017% I-chloro-lnaphthol in Tris saline.

Shielding
of Cytochrome bs at the Stromal Surface-Although the protease carboxypeptidase A by itself has no effect on cytochrome bs (Fig. 2 Thylakoids were incubated with carboxypeptidase A for 15 min, then with trypsin for 7.5 min at room temperature: lane a, control not treated with protease: lane b, trypsintreated thylakoids (1:20 (w/w), trypsin:chlorophyll); lanes c and d, sequential addition to the thylakoids of carboxypeptidase A (1:8 (c), 1:l (d), w/w, carboxypeptidase Achlorophyll, respectively), and then trypsin (1:20 w/w) after carboxypeptidase A action was terminated by o-phenanthroline; lane e, carboxypeptidase A-treated thylakoids (1:l (w/w), carboxypeptidase A:chlorophyll).
Other conditions as under "Materials and Methods." A, immunodetection of proteolysis of cytochrome bs using antibody against the COOH-terminal decapeptide, Ile-205 to Leu-214. B, immunodetection of proteolysis of cytochrome bg using antibody against the NHP-terminal decapeptide, Asp-5 to Gln-14. Other conditions as in A. to thylakoid membranes was increased if the thylakoids were treated first with carboxypeptidase A. As seen by antibody (Western) analysis, cleavage of cytochrome bs by trypsin to a slightly smaller protein (AitfI = -l,OOO-2,000; M, = 21,000-22,000 of cleaved product) occurred more completely if the membranes had been pretreated with the carboxypeptidase (compare lanes c and d in Fig. 2 to lane b). o-Phenanthroline was added to inhibit the carboxypeptidase before trypsin treatment so that the two proteases did not act simultaneously. When the order of protease addition was reversed, with trypsin added first and inhibited by phenylmethylsulfonyl fluoride before addition of carboxypeptidase, there was no increase in the extent of proteolysis by trypsin. Therefore, the access of trypsin to the terminal epitopes of cytochrome bs was increased by prior treatment with carboxypeptidase. Thus, the segments of cytochrome bs that are exposed on the stromal membrane surface, including the NH*-and COOHterminal segements, are shielded by the COOH termini of other thylakoid proteins. A similar effect of carboxypeptidase on the trypsin accessibility to cytochrome bs was observed previously using heme stain for detection of cytochromes (Szczepaniak et al., 1989). The COOH terminus of cytochrome f in the bcf complex is cleaved by carboxypeptidase A added to thylakoids, is exposed on the stromal surface (Willey et al., 1984), and could be involved in shielding cytochrome be.
Independently of the orientation of the COOH terminus of cytochrome bg in the membrane, the insensitivity of cytochrome bs to carboxypeptidase A is not unexpected, since the proline residue at the penultimate position of cytochrome bs should block the action of the carboxypeptidase (Ambler, 1967), and an earlier report to the contrary was probably due to a contaminating protease in the carboxypeptidase (e.g. chymotrypsin; Ambler, 1967).
The band seen at slightly smaller M, values (aM, = -l,OOO-2,000) in a Western blot using the antibody against the COOH-terminal and of cytochrome bs (Fig. 211) must arise from cleavage at the NH, terminus. Although the NH,-terminal epitope appears to be slightly more sensitive to trypsin than the COOH-terminal epitope in these experiments, as judged by the presence of the band running at a slightly smaller M, value in Fig. 24 compared to its absence in Fig.  2B (antibody to NH, epitope), the difference in sensitivity of the NH2 and COOH termini must be small because it was not seen in all experiments (see below, Fig. 5).

Relative Rate of Trypsin Proteolysis of Cytochrome bs in
Thylukoids-The accessibility/sensitivity to trypsin of epitape(s) III of the antibody to the entire cytochrome b6, and epitopes I and II near the NH, and the COOH termini, were compared in thylakoid membranes to that of epitopes of cytochrome f (Figs. 3 and 4) and the OEC extrinsic polypeptides (Figs. 5-7) located on the lumen side of the membrane.
Most of the cytochrome f polypeptide, including all of its trypsin-sensitive sites, is also located on the lumen side of the membrane (Willey et al., 1984;Szczepaniak et al., 1989). Fig. 3, A-D, lanes b-i, show the effect in one experiment, using membranes pretreated with carboxypeptidase, of increasing trypsin:chlorophyll ratios and times of incubation on the integrity of cytochrome f (Fig. 3A), the COOH-and NH*terminal epitopes II and I of cytochrome bs (Fig 3, B and C), and epitope(s) III of cytochrome bs (Fig. 30). The greater sensitivity to trypsin of all of the cytochrome b6 epitopes can be seen. A graph of the undigested epitopes as a function of the trypsin:chlorophyll ratio for several experiments is shown in Fig. 4. The data shown in Fig. 4 did not involve membranes pretreated with carboxypeptidase, but this did not affect the relative trypsin sensitivity of the different epitopes compared The thylakoids were then incubated with trypsin for 7.5 min (l:lOO, 1:40, 1:20, 1:lO (w/w), trypsin:chlorophyll, lanes c, d, e, and f) at room temperature. Lane a, control; lane b, carboxypeptidase A-treated thvlakoids. Membrane nroteins were senarated in 15% SDS-PAGE and cytochrome f was detected by Western blotting. Each lane was loaded with the membrane equivalent of 10 pg of chlorophyll. B,immunodetection of cytochrome be trypsinolysis using antibody against epitope II, the COOH-terminal decapeptide, Ile-205 to Leu-214. Conditions as in A, except that the Western blot used the peptide-directed antibody. C, immunodetection of cytochrome bg trypsinolysis using antibody against NHB-terminal decapeptide, epitope I, Asp-5 to Gln-14. Thylakoids were incubated with trypsin for 7.5 min (l:lOO, 1:40, 1:20, 1:lO (w/w), trypsin:chlorophyll, lanes b, c, d, and e) at room temperature. Lane a, control. Carboxypeptidase A-treated sample not shown. Other conditions as in A, except that the Western blot used the peptide-directed antibody. D, immunodetection of cytochrome be trypsinolysis using antibody III to the entire protein. to each other. The values plotted for the undigested COOHterminal epitope II and epitope(s) III of the whole protein are those of the band at the unshifted position of the control in the gel, as shown in Fig. 3, B and D. The data in this experiment also indicate that (i) the virtually simultaneous cleavage (AMr = -2,OOO-3,000) of both NH2 and COOH termini by trypsin can be detected with antibody III to the intact protein (Figs. 30 and 50; Table I); the loss of the NH, epitope with increasing exposure to trypsin (Figs. 2B and 3C), without concomitant observation of a slightly smaller band, indicates that one does not observe cleavage of the COOH terminus alone. (ii) A small fraction of the cytochrome bs population starting in lone e (trypsin:chlorophyll = 0.05) of Fig. 3B shows cleavage of the NH, terminus alone. The AM, value of -2,OOO-3,000 for the slightly smaller component (Mr 21,000) generated by trypsinolysis that reacts with antibody III to the intact cytochrome (Fig. 30) is approximately twice that for antibody II to the COOH-terminal epitope (Fig. 3B), implying that antibody III detects cytochrome bs cleaved simultaneously at both termini. Antibody III must then react with at least one epitope that is resistant to the trypsin added to thylakoids, in addition to reacting with the NH*-and COOH-terminal epitopes. The presence of lower molecular size bands in these gels due to proteolysis at positions within the protein might have been anticipated. Such bands of M, > 3000-4000 could have been detected, but were not found. The absence of such proteolytic cleavage products was noted previously in a study of bacteriorhodopsin proteolysis, and was attributed to destabilization by protease treatment of the membrane-embedded segments that allowed further access to protease and extensive proteolysis (Dumont et al., 1985).  Fig. 3 except membranes were not pretreated with carboxypeptidase A. The 16-kDa polypeptide was detected by a Western blot using antibody to the entire protein. B, immunodetection of cytochrome bs trypsinolysis using antibody against the COOH terminus decapeptide, Ile-205 to Leu-214. Conditions as in A, except that the Western blot used the peptide-directed antibody. C, immunodetection of cytochrome b6 trypsinolysis using antibody against NH*-terminal decapeptide, Asp-5 to Glu-14. Conditions as in A, except that the Western blot used the peptide-directed antibody. D, trypsin proteolysis of cytochrome bs. Conditions as in A except that the cytochrome bs was detected by Western blots using antibody to the entire protein.  The 16-kDa OEC polypeptide was also found to be relatively resistant to trypsin added to thylakoid membranes compared to epitopes I-III of cytochrome bs (15-min digestion, no carboxypeptidase A pretreatment) (Figs. 5 and 6). Trypsinolysis of epitope(s) III of cytochrome bs again results in a slightly smaller M, = 21,000 product with the sum of the density of the protein bands in lanes b-f (Fig. 50) equal to 85-90% of the control (Fig. 5D, lane a) (Table I), supporting the conclusion that the nli, 21,000 band arose from simultaneous cleavage at both NH2 and COOH termini. Thus, the loss of the M, 23,000 cytochrome bs caused by trypsinolysis of thylakoid membranes is caused by cleavage at the NH, and COOH termini and not to cleavage at another exposed site within cytochrome be NH2 and COOH terminal epitopes, and 16-kDa OEC extrinsic protein to the trypsin added from the stromal side using mostly unstacked thylakoids, derived from the data shown in Fig. 5. Relative protein concentrations were measured by densitometric analysis of the Western blot. The points are expressed as percentages of the control sample and are the means of four to five different preparations. Data from lanes g and h were not plotted.
Epitopes OE A, 16-kDa OEC extrinsic protein; n , NH, terminus of cytochrome b6; standard deviations representative of all bG samples are shown; 0, COOH terminus of cytochrome bs; A, entire cytochrome bs.
The relative rate of cleavage of the M, 33,000 OEC polypeptide by trypsin also indicated a smaller accessibility of this OEC protein to trypsin relative to the cytochrome bs epitopes I-III, as summarized in the graph of Fig. 7.
The sensitivity/accessibility of the NH2-and COOH-terminal epitopes of cytochrome bs to trypsin, relative to the OEC polypeptides, is reversed in inside-out membranes (Figs. 8 and 9), compared to the intact thylakoids. The relative sensitivities to trypsin are (Fig. 8): cytochrome bs (NH*) (epitope I) = cytochrome bs (COOH) (epitope II) < 33 kDa < 16 kDa. The relative sensitivity of the 16-and 33-kDa polypeptides to each other in the inner membrane surface is as expected from their extractability from inside-out membranes and their relative proximity to the membrane-bound manganese of photosystem II (Kuwabara and Murata, 1983;Seibert et al., 1987). The smaller relative sensitivity of the cytochrome bs NH, and COOH epitopes I and II is observed in spite of a greater absolute sensitivity of the cytochrome bs epitopes to trypsin in the inside-out thylakoid membranes (Fig. 9). The greater absolute sensitivity of cytochrome bs detected by antibodies I and II would be expected if trypsinsensitive sites (K-148, R-169) in the peptide loop between helices III and IV are exposed on the lumen side of the membrane, as predicted by the 4-helix model (Fig. 1A). This would not be expected from a 5-helix model (Fig. 1B) because in the latter model there are no trypsin sites on the lumen side. The absence of lower molecular weight products in these gels again implies that cytochrome bs may be destabilized in the membrane after initial proteolysis at an internal exposed Conditions as in Fig. 5. The points are the means of three different experiments. Epitopes of: 0,33-kDa OEC extrinsic protein; n , NH, terminus of cytochrome bs; 0, COOH terminus of cytochrome bs; and A, entire protein of cytochrome b6; standard deviation representative of all cytochrome bg samples is shown.
loop, allowing further access to protease and extensive degradation (Dumont et al., 1985). DISCUSSION The relative sensitivity of epitopes of cytochrome b6 and of other thylakoid membrane proteins to trypsin was used in the present work as an indicator of their sidedness and orientation in the membrane bilayer. The net sensitivity of an epitope will depend not only on its position in the membrane, but also on its intrinsic sensitivity (i.e. in the case of trypsin, number of accessible lysine and arginine residues), and on the extent of the shielding of these residues by other intrinsic and peripheral membrane proteins and the membrane surface. The shielding of cytochrome bs by the COOH termini of other proteins demonstrated here and in Szczepaniak et al. (1989) is an example of the latter effect. The OEC proteins, particularly the 16-kDa polypeptide (very sensitive) and cytochrome f (relatively insensitive) represent two opposite ends of the spectrum with respect to their sensitivity to trypsin on the lumen side of the membrane. The relative insensitivity of cytochromefto V8 protease, compared to the exposed lumen side loops of cytochrome bs was documented previously (Szczepaniak et al., 1989). The high intrinsic sensitivity of the OEC proteins to trypsin (Isogai et al., 1985;Jansen et al., 1987;Tae and Cramer, 1989) suggests that they would be expected to be more sensitive to trypsin than cytochrome f, and perhaps the most trypsin-sensitive of the proteins on the lumen side.
The dependence of each of the epitope or epitope systems of cytochrome bs and the reference proteins on the amount of trypsin added to thylakoids is not a single monophasic function (Figs. 4,6, and 7). These graphs indicate that 30-50% of these epitopes are a relatively resistant fraction. At least part of the resistant fraction might be attributed to stacking of the membranes, because approximately 25% of the thylakoids were stacked ("Materials and Methods").
The comparison of the susceptibility of the NH*-and COOH-terminal epitopes to trypsin in the intact thylakoids (order of susceptibility: cytochrome f < 16 kDa = 33 kDa C COOH-cytochrome bs I NH*-cytochrome be), with that in inside-out thylakoid membranes (order of susceptibility: NH*cytochrome b6 = COOH-cytochrome bs < 33 kDa = 16 kDa), implies that both the NH2 and COOH termini are on the stromal side of the membrane. This orientation is also implied by the comparison of the tryptic cleavage products of cytochrome bs by epitopes I, II, and epitope(s) III, indicating that this cleavage occurs predominantly by simultaneous removal of both termini. The number of membrane-spanning helices is therefore even and most likely four.
The main features of the 4-helix model are that (i) the two hemes cross-link helices II and IV, the heme on the lumen side (b,) coordinated by His-85 (helix II) and His-186 (helix II), and that on the stromal side (heme b,) by His-99 (helix II) and His-201 (helix IV). (ii) The 2 His residues on helix II are separated by 13 residues, and the 2 on helix IV by 14. The inter-His distance on helix IV is one more than in cytochrome b(bcJ and has been proposed to account for the more homogeneous redox and spectral properties of the two hemes of cytochrome bs ). An additional unique intramembrane property of cytochrome bs is the arginine residue at position 86 in the bilayer next to His-85. This residue is apparently conserved in the chloroplast and cyanobacterial sequences, but not in cytochrome b(bq), and is the only charged residue of cytochrome bs that appears to be in the hydrophobic membrane bilayer. (iii) Fairly long peripheral loops (about 25 and 42 residues, respectively) connect helices I-II and III-IV, on the lumen side of the membrane. The latter loop is analogous to a region of the cytochrome b(bcl) of Rb. capsulatus that has been inferred from the presence of a large number ofp-side inhibitor-resistant loci, to be involved in quinone binding (Daldal et al., 1989). This is consistent with the model of Brasseur (1988) in which this peptide segment is proposed to be an amphipathic a-helix bound closely to the membrane surface. On the other hand, this III-IV interhelix loop was found to be much more accessible to V8 protease in permeabilized thylakoids than was cytochrome f (Szczepaniak and Cramer, 1989), implying that the interhelix loop III-IV of cytochrome bs protrudes from the membrane surface. In addition, the binding of quinone photoaffinity labels to subunit IV and not cytochrome bs indicates that the former polypeptide and not the latter binds the quinol donor to the bGf complex (Doyle et al., 1989). In this case, the III-IV interhelix loop of cytochrome bs, unlike that of cytochrome b(bc,), would not be involved in the quinone binding function. The trypsin probe experiments of cytochrome bs in the thylakoid membrane reported here indicate that in terms of orientation, topography, and heme binding, cytochrome bs is analogous to the heme-binding domain contained in the NHz-terminal half of the larger cytochrome b of the cytochrome bcl complex.