TIBS 15-AUGUST 1990

JOURNALCLUB The cytochrome bc~ complexes of photosynthetic purple bacteria play a key role in light-driven, energy-transducing cyclic electron flow~-6 and also participate in respiratory electron flow during aerobic growth 7. Similar complexes are found in the mitochondrial membranes of eukaryotic aerobes L2,s and the related cytochrome bJ complex is found in the membranes of oxygenic phototrophs 1-t. The photosynthetic bacterial cytochrome bc~ complexes catalyse electron flow from ubiquinol to cytochrome c2 and utilize the energy released to create a transmembrane electrical potential and a pH gradient, which can subsequently be used as an energy source for ATP synthesis ~-6. These complexes all contain a core of three peptides that carry a total of four electron transferring prosthetic groups: The Rieske iron-sulfur protein, which contains a single [2Fe-2S] cluster; cytochrome c~, which contains a single covalently bound heme c, and cytochrome b, which contains two noncovalently bound protohemes that have different absorbance spectra and Em values~-E The cytochrome bcl complexes of photosynthetic bacteria have two important experimental advantages when compared to the corresponding mitochondrial complexes: (1) the progress of single electrons through the complexes can be followed using short, single-turnover light pulses to initiate electron flowL3'4; and (2) the peptide subunit composition of these bacterial complexes is much simpler than those of the corresponding mitochondrial complexes 1,5,6,e-~°(see also H. Weiss et al. [1990] TIBS 15, 178-180). Recent research on the cytochrome bc, complexes of photosynthetic bacteria has provided more information on the peptide composition u-13 and gene sequences of the complex 14'~s. New evidence has also emerged on the likely folding pattern of the cytochrome b peptide ~6,on the identity of iron-binding iigands ~7J8 and on the identity of electrogenic electron transfer steps within the complex xg.

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peptides that have electron-carrying prosthetic groups, the possibility exists that some of them may contain a fourth subunit. There has been recent confirmation u'~2 of earlier reports ~°,2°that the Rhodobacter sphaeroides cytochrome bc~ complex contains a subunit with molecular mass of approximately 14 kDa, in addition to cytochrome b, cytochrome c~ and the Rieske ironsulfur protein. Confirmation has also been obtained u that the RhodospiriUum rubrum complex has only three subunits and no 14 kDa subunit ~3,2~,and the same would appear to be true for the Rhodopseudomonas viridis cytochrome bc~ complexg.2L There is some uncertainty as to whether the complex from a fourth photosynthetic bacterium, Rhodobacter capsulatus, contains three or four subunits 9,~°. It is possible that the 14 kDa subunit observed in some preparations is a fortuitous contaminant. Alternatively, all these complexes may contain four subunits in situ and the 14 kDa subunit may be lost from some species during purification. Of course, the subunit composition may differ in different species. A partial amino acid sequence obtained for the 14 kDa peptide found in the Rb. sphaeroides preparation revealed no significant homology to any known proreins u, nor provided any support for the postulated role of this subunit as a quinone-binding protein 22. It appears that the role of the putative fourth subunit of the cytochrome bc~ complexes of photosynthetic bacteria may be best determined by genetic techniques, once the gene for this protein has been identified. Until recently, there had been gene sequences available only for the three electron transfer subunits of the Rb. capsulatus complex23,24. Sequences Subunitcomposition Although it is clear that these bac- are now also available for these terial complexes contain only three peptides from Rps. viridis~4 and Rbo

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sphaeroideslE The availability of these genes and knowledge of their sequences will allow analyses of the importance of specific amino acids for the functions of the complex through comparisons of conserved amino acid residues and through site-specific mutagenesis experiments.

Foldingof cytochromeb Figure 1 shows a model for the likely folding, in the plasma membrane, of the cytochrome b peptide of the Rb. capsulatus cytochrome bcl complex~E Earlier models, based largely on hydropathy plots, contained nine transmembrane helices25.2E The new model, which eliminates the least hydrophobic helix of the earlier models, incorporates information derived from sequencing the cytochrome b gene in Rb. capsulatus mutants that are resistant to several specific inhibitors of the cytochrome bc~ complex~E This eight-transmembrane helix model has the advantage of placing amino acids implicated in binding the same inhibitor close to one another, while the earlier (nine-transmembrane helix) model placed some of these residues on different sides of the membrane '6,27,28. Similar models have also been proposed from studies of inhibitor-resistance mutations in yeast cytochrome b 27,2s.The location of these yeast mutations, together with inhibitor-resistant mutations in mouse mitochrondrial cytochrome b29, are also shown in Fig. 1.

Iron IIgands Both the eight- and nine-transmembrahe helix models identify four histidine residues that are conserved in all known cytochrome b sequences as axial ligands to the two protoheme irons of cytochrome b 9,16,25-31.Electron paramagnetic resonance (EPR) studies of mitochondrial cytochrome b32, chloroplast cytochrome b633, and of model compounds 32 had been interpreted as evidence for the presence of two histidine axial ligands for each of the cytochrome b heine irons, but the unusual low-field g values for cytochrome b had to be rationalized in terms of 'strained' conformations 3L32. New near-infrared magnetic circular dichroism studies have 289

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provided additional evidence of bishistidyl axial ligation for the two protoheme groups of mitochondrial cytochrome b and also established that the likely axial ligands for the heine iron of cytochrome c~ are methionine and histidine a4. The absolute conservation of four specific histidine residues in the amino acid sequences of cytochromes b from mitochrondria and photosynthetic bacteria 9J4-16.23'24,a° strongly suggested that bis-histidyl ligation for the cytochrome b hemes also occurs in photosynthetic bacteria, but direct evidence on this question has been absent until now. Resonance Raman spectroscopic measurements on the R. rubrum cytochrome bq complex have now provided confirmation of this bis-histidyl axial ligation for the cytochrome b hemes and of the likely presence of histidine and methionine as axial ligands for cytochrome c, '7. An additional similarity between the mitochondrial, chloroplast and photosynthetic bacterial cytochrome bc, complexes was demonstrated in an electron spin echo envelope modulation (ESEE1V0 spectroscopic investigation of the [2Fe-2S] cluster of the Rieske iron-sulfur proteins of these 290

complexes. The ESEEM spectra of the Rieske iron-sulfur proteins from the chloroplast cytochrome bJ complex, the beef heart mitochondrial cytochrome bc, complex and complexes from two photosynthetic bacteria (R. rubrum and Rb. sphaeroides) were all quite similar and suggested the presence of two nitrogen ligands, presumably from conserved histidines, to the irons of the cluster TM.

Electrogenic electron transfer steps So-called Q-cycle models ~-~ for electron flow through the cytochrome bc, complex feature electron transfer from the lower-potential heme (bL) of cytochrome b, located between histidines 97 and 198, to the higher-potential heme (bH), located between histidines 111 and 212. The structural model of Fig. 1 places these two hemes at different distances from the interfaces between the membrane lipid bilayer and the aqueous phases and thus predicts that electron transfer from b c to bH should be electrogenic (i.e. should result in net charge separation across the membrane). This was originally demonstrated some time ago for the Rb. sphaeroides complexss and

more recent studies have provided a complete picture of all the likely electrogenic steps within the complex ~9. Shifts in the absorbance spectrum of membrane-bound carotenoids that result from the formation of a transmembrane electrical potential have been used to identify the steps during electron flow through the complex that are electrogenic. Electron flow from cytochrome bL to bH accounts for approximately 60% of the transmembrahe charge separation, with the remaining portion of the membrane potential arising from electron flow between reduced cytochrome bL and an oxidized ubiquinone, which becomes reduced at the so-called Q¢ site (see Fig. 1). The results of this study indicate that the hemes of cytochromes c, and bLand the [2FeZ2S] cluster of the Rieske iron-sulfur protein are located out of the low dielectric portion of the membrane on the periplasmic side and that the bH heme is buried in the low dielectric portion of the membrane '9.

References I Hauska, G., Hurt, E., Gabellini, N. and Lockau, W. (1983) Biochim. Biophys. Acta 726, 97-133 2 Rich, P. R. (1984) Biochim. Biophys. Acta 768, 53--79 3 Crofts, A. R. and Wraight, C. A. (1983) Biochim. Biophys. Acta 726, 149-185 4 Dutton, P. L. (1986) in Encyclopedia of Plant Physiology (Staehlin, L. A. and Amtzen, C. A., eds), New Series Vol. 19, pp. 197-237, Springer-Verlag 5 Cramer, W. A., Black, M. T., Widger, W. R. and Girvin, M. E. (1987) in The Light Reactions (Barber, J., ed.), pp. 447-493, Elsevier 6 Malkin, R. (1988) in ISl Atlas of Science: Biochemistry, pp. 57-64 7 Zannoni, D. and Melandri, B. A. (1985) in Coenzyme Q. Biochemistry, Bioenergetics and Clinical Applications of Ubiquinone (Lenaz, G., ed.), pp. 235-265, John Wiley & Sons 8 Yang, X., Ljungdahl, P. 0., Payne, W. E. and Trumpower, B. L. (1987)in Bioenergetics: Structure and Function of Energy Transducing Systems (Ozawa,T. and Papa, S., eds), pp. 63-80, Japan Sci. Press, Tokyo/Springer-Verlag 9 Gabellini, N. (1988) J. Bioenerg. Biomembr. 20, 59-83 10 Ljungdahl, P. 0., Pennoyer,J. D., Robertson, D. E. and Trumpower, B. L. (1987) Biochem. Biophys. Acta 891, 227-241 11 Purvis, D. J., Theiler, R. and Niederman, R. A. (1990) J. Biol. Chem. 265, 1208-1215 12 Andrews, K. M., Crofts, A. R. and Gennis, R. B. (1990) Biochemistry 29, 2645-2651 13 Kriauciunas, A., Yu, L., Yu, C-A., Wynn, R. M. and Knaff, D. B. (1989) Biochim. Biophys. Acta 976, 70-76 14 Verbist, J., Lang, F., Gebellini, N. and Oesterhelt, D. (1989) Mol. Gen. Genet. 219, 445-452 15 Yun, C-H., Beci, R., Crofts, A. R. and Gennis, R. B. Eur. J. Biochem. (submitted) 16 Dalclal, F., Tokito, M. K., Davidson, E. and Faharn, M. (1989) EMBO J. 13, 3951-3961

TIBS 1 5 - A U G U S T 1 9 9 0 17 Hobbs, J. D., Kriauciunas,A., G(Jner,S., Knaff, D. B. and Ondrias, M. O. Biochim. Biophys. Acta (in press) 18 Britt, R. D., Sauer, K., Klein, M. P,, Knaff, D. B.,

Kriauciunas,A., Yu, C-A.,Yu, L. and Malkin, R. Biochemistry (submitted) 19 Robertson, D. E. and Dutton, P. L. (1988) Biochim. Biophys. Acta 935, 273-291 20 Yu, L., Mei Q-C.and Yu, C-A.(1984) J. Biol. Chem. 259, 5752-5760 21 Wynn, R. M., Gaul, D. F., Choi, W-K., Shaw, R. W. and Knaff, D. B. (1986) Photosynth. Res. 9, 181-195 22 Yu, L. and Yu, C-A.(1987) Biochemistry 26, 3658-3664 23 Davidson,E. and Daldal, F. (1987) J. Mol. Biol.

ALTHOUGH MUCH IS KNOWN about the structure and biosynthesis of the oligosaccharides in glycoproteins, the central question of how glycosylation contributes to glycoprotein structure and function remains unclear. Functional roles for carbohydrate that have been identified include targeting of lysosomal enzymes, inducing appropriate folding patterns for the nascent polypeptide, and providing specific carbohydrate structures that participate in biological recognition ]. Each of these functions, however, applies to a few glycoproteins or to a specific class of glycoproteins but none of them represents a universal role for glycosylation. In fact, for many glycoproteins it is not clear whether the carbohydrate serves any function at all. This is particularly true for glycoproteins with O-linked oligosaccharides since these have been less intensively studied than the more common glycoproteins with N-linked sugars. For O-linked sugars, there is evidence for participation in biological phenomena such as cell-cell interactions (reviewed in Refs 1 and 2). Similarly, activation of T-lymphocytes is associated with substantial changes in the carbohydrate structure of leukosialin, the major surface glycoprotein in these cells3 and this ability to alter carbohydrate structure may be required for proper T-cell function 4. However, despite their intrinsic N. Jentoftis at the Departments of Pediatrics and Biochemistry, Case Western Reserve University, Cleveland, OH 44106, USA.

195, 13-24 24 Gabellini,N. and Sebald,W. (1986) Eur. J. Biochem. 154, 569-579 25 Saraste, M. (1984) FEBS Lett. 166, 367-372 26 Widger, W. R., Cramer,W. A., Herrmann, R. G. and Trebst, A. (1984) Proc. Natl Acad. Sci. USA

81, 674-678 27 di Rago,J. P. and Colson, A-M. (1988) J. Biol. Chem. 263, 12564-12570 28 Brasseur, R. (1988) J. Biol. Chem. 263,

977, 249-265 32 Palmer,G. (1985) Trans. Biochem. Soc. 13,

548-560 33 Nitschke, W. and Hauska,G. (1987) FEBS Lett.

213, 453-455 34 Simpkin, D., Palmer,G., Devlin,F. J., McKenna,

M. C., Jensen, G. M. and Stephens, P. J. (1989) Biochemistry 28, 8033-8039 35 Glaser, E. and Crofts, A. R. (1984) Biochim. Biophys. Acta 935, 273-291

12571-12575 29 Howell,N. and Gilbert, K. (1988) J. Mol. Biol.

203, 607-618 30 Hauska,G., Nitschke,W. and Herrmann, R. G. (1988) J. Bioenerg. Biomembr. 20, 211-228 31 Degli Eposti, M. (1989) Biochim. Biophys. Acta

DAVID B. KNAFF Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA.

The O-linked oligosaccharides of glycoproteins are usually clustered within heavily glycosylated regions of the peptide chain. Steric interactions between carbohydrate and peptide within these clusters induce the peptide core to adopt a stiff and extended conformation and this conformational effect appears to represent a major function of O-glycosylation.

interest, these biological roles have been shown to apply only to a few glycoproteins. This article will focus on the effects of Oglycosylation that are likely to be valid for all glycoproteins containing these structures. One apparently universal consequence of O-glycosylation is the relative resistance to proteases of O-glycosylated regions in glycoproteins. This is frequently exploited to generate very large (300-400 kDa) glycopeptides from mucous glycoproteins (mucins) 5 or smaller glycopeptides from membrane glycoproteins ~,7. The most likely explanation for protease resistance is simply that the attached carbohydrate blocks access to the peptide core since these same sequences are quite susceptible to proteases in the absence of attached carbohydrate s. The second consequence of O-glycosylation and the primary subject of this review is the induction of a specific conformation. Although O-linked sugars are occasionally found in serum glycoproreins, are present in certain nuclear glycoproteins9, and are fairly common

© 1990,ElsevierSciencePublishersLtd,(UK) 0376-5067/90/$02.00

in viral glycoproteins, they are more usually thought of as constituents of cellsurface glycoproteins and mucins. The carbohydrate chains in these glycoproteins range in size from one to more than 20 sugars with the larger oligosaccharides displaying considerable structural and antigenic diversity l°'u. These oligosaccharides are not uniformly distributed along the peptide chain; typically they are clustered in heavily glycosylated domains in which glycosylated serine and threonine residues comprise 25--40% of the sequence 5'7'12-]4. In mucins, these heavily glycosylated sequences are about 600-1200 amino acids long and appear to be separated by much shorter nonglycosylated regions s, whereas the heavily glycosylated domains in other glycoproteins are much smaller, often consisting of 20-70 amino acids 7,]2-]4.Since the carbohydrate content in these heavily glycosylated regions ranges from 65-85%, it dominates their chemical and physical properties. Secondary and tertiary structures that are typical of unsubstituted peptide chains are precluded by 291