William A. Cramer

Crystal structure of chloroplast cytochrome freveals a novel cytochrome fold and unexpected heme ligation

BACKGROUND Cytochrome f is the high potential electron acceptor of the chloroplast cytochrome b6f complex, and is the electron donor to plastocyanin. The 285-residue cytochrome f subunit is anchored in the thylakoid membrane of the chloroplast by a single membrane-spanning segment near the carboxyl terminus. A soluble redox-active 252-residue lumen-side polypeptide with native spectroscopic and redox properties, missing the membrane anchor and carboxyl terminus, was purified from turnip chloroplasts for structural studies. RESULTS The crystal structure of cytochrome f, determined to 2.3 A resolution, has several unexpected features. The 252-residue polypeptide is organized into one large and one small domain. The larger heme-binding domain is strikingly different from known structures of other c-type cytochromes and has the same fold as the type III domain of the animal protein, fibronectin. Cytochrome f binds heme with an unprecedented axial heme iron ligand: the amino terminus of the polypeptide. CONCLUSION The first atomic structure of a subunit of either the cytochrome b6f complex or of the related cytochrome bc1 complex has been obtained. The structure of cytochrome f allows prediction of the approximate docking site of plastocyanin and should allow systematic studies of the mechanism of intra- and inter-protein electron transfer between the cytochrome heme and plastocyanin copper, which are approximately isopotential. The unprecedented axial heme iron ligand also provides information on the sequence of events (i.e. cleavage of signal peptide and ligation of heme) associated with translocation of the cytochrome across the membrane and its subsequent folding.

Full Subunit Coverage Liquid Chromatography Electrospray Ionization Mass Spectrometry (LCMS+) of an Oligomeric Membrane Protein Cytochrome b6f Complex From Spinach and the Cyanobacterium Mastigocladus Laminosus

Highly active cytochrome b6f complexes from spinach and the cyanobacterium Mastigocladus laminosus have been analyzed by liquid chromatography with electrospray ionization mass spectrometry (LCMS+). Both size-exclusion and reverse-phase separations were used to separate protein subunits allowing measurement of their molecular masses to an accuracy exceeding 0.01% (±3 Da at 30,000 Da). The products of petA, petB, petC, petD, petG, petL, petM, and petN were detected in complexes from both spinach and M. laminosus, while the spinach complex also contained ferredoxin-NADP+ oxidoreductase (Zhang, H., Whitelegge, J. P., and Cramer, W. A. (2001) Flavonucleotide:ferredoxin reductase is a subunit of the plant cytochrome b6f complex. J. Biol. Chem. 276, 38159–38165). While the measured masses of PetC and PetD (18935.8 and 17311.8 Da, respectively) from spinach are consistent with the published primary structure, the measured masses of cytochrome f (31934.7 Da, PetA) and cytochrome b (24886.9 Da, PetB) modestly deviate from values calculated based upon genomic sequence and known post-translational modifications. The low molecular weight protein subunits have been sequenced using tandem mass spectrometry (MSMS) without prior cleavage. Sequences derived from the MSMS spectra of these intact membrane proteins in the range of 3.2–4.2 kDa were compared with translations of genomic DNA sequence where available. Products of the spinach chloroplast genome, PetG, PetL, and PetN, all retained their initiating formylmethionine, while the nuclear encoded PetM was cleaved after import from the cytoplasm. While the sequences of PetG and PetN revealed no discrepancy with translations of the spinach chloroplast genome, Phe was detected at position 2 of PetL. The spinach chloroplast genome reports a codon for Ser at position 2 implying the presence of a DNA sequencing error or a previously undiscovered RNA editing event. Clearly, complete annotation of genomic data requires detailed expression measurements of primary structure by mass spectrometry. Full subunit coverage of an oligomeric intrinsic membrane protein complex by LCMS+ presents a new facet to intact mass proteomics.

The structure of BtuB with bound colicin E3 R-domain implies a translocon

Cellular import of colicin E3 is initiated by the Escherichia coli outer membrane cobalamin transporter, BtuB. The 135-residue 100-Å coiled-coil receptor-binding domain (R135) of colicin E3 forms a 1:1 complex with BtuB whose structure at a resolution of 2.75 Å is reported. Binding of R135 to the BtuB extracellular surface (ΔG° = −12 kcal mol−1) is mediated by 27 residues of R135 near the coiled-coil apex. Formation of the R135–BtuB complex results in unfolding of R135 N- and C-terminal ends, inferred to be important for unfolding of the colicin T-domain. Small conformational changes occur in the BtuB cork and barrel domains but are insufficient to form a translocation channel. The absence of a channel and the peripheral binding of R135 imply that BtuB serves to bind the colicin, and that the coiled-coil delivers the colicin to a neighboring outer membrane protein for translocation, thus forming a colicin translocon. The translocator was concluded to be OmpF from the occlusion of OmpF channels by colicin E3.

On the structure and function of cytochrome b-559.

A sumary of biochemical, biophysical, and molecular biological data is presented which led to the identification of two different polypeptides (α and β, MW=9.16 and 4.27 kDa) in the cytochrome b-559 protein. The presence of a single His residue on each polypeptide, and the conclusion from spectroscopy that the heme coordination must be bis-histidine led to an obligatory requirement for coordination of a single heme through a heme cross-linked dimer. This structure does not have a precedent among soluble or membrane bound cytochromes. The possible participation of the cytochrome in the pathway of photoactivation is discussed.

Crystal structures of the OmpF porin: function in a colicin translocon

The OmpF porin in the Escherichia coli outer membrane (OM) is required for the cytotoxic action of group A colicins, which are proposed to insert their translocation and active domains through OmpF pores. A crystal structure was sought of OmpF with an inserted colicin segment. A 1.6 Å OmpF structure, obtained from crystals formed in 1 M Mg2+, has one Mg2+ bound in the selectivity filter between Asp113 and Glu117 of loop 3. Co‐crystallization of OmpF with the unfolded 83 residue glycine‐rich N‐terminal segment of colicin E3 (T83) that occludes OmpF ion channels yielded a 3.0 Å structure with inserted T83, which was obtained without Mg2+ as was T83 binding to OmpF. The incremental electron density could be modelled as an extended poly‐glycine peptide of at least seven residues. It overlapped the Mg2+ binding site obtained without T83, explaining the absence of peptide binding in the presence of Mg2+. Involvement of OmpF in colicin passage through the OM was further documented by immuno‐extraction of an OM complex, the colicin translocon, consisting of colicin E3, BtuB and OmpF.

A defined protein–detergent–lipid complex for crystallization of integral membrane proteins: The cytochrome b6f complex of oxygenic photosynthesis

The paucity of integral membrane protein structures creates a major bioinformatics gap, whose origin is the difficulty of crystallizing these detergent-solubilized proteins. The problem is particularly formidable for hetero-oligomeric integral membrane proteins, where crystallization is impeded by the heterogeneity and instability of the protein subunits and the small lateral pressure imposed by the detergent micelle envelope that surrounds the hydrophobic domain. In studies of the hetero (eight subunit)-dimeric 220,000 molecular weight cytochrome b6f complex, derived from the thermophilic cyanobacterium, Mastigocladus laminosus, crystals of the complex in an intact state could not be obtained from highly purified delipidated complex despite exhaustive screening. Crystals of proteolyzed complex could be obtained that grew very slowly and diffracted poorly. Addition to the purified lipid-depleted complex of a small amount of synthetic nonnative lipid, dioleolyl-phosphatidylcholine, resulted in a dramatic improvement in crystallization efficiency. Large crystals of the intact complex grew overnight, whose diffraction parameters are as follows: 94% complete at 3.40 Å spacing; Rmerge = 8.8% (38.5%), space group, P6122; and unit cell parameters, a = b = 156.3 Å, c = 364.0 Å, α = β = 90o, γ = 120o. It is proposed that the methodology of augmentation of a well-defined lipid-depleted integral membrane protein complex with synthetic nonnative lipid, which can provide conformational stability to the protein complex, may be of general use in the crystallization of integral membrane proteins.

STOICHIOMETRICALLY BOUND BETA -CAROTENE IN THE CYTOCHROME B6F COMPLEX OF OXYGENIC PHOTOSYNTHESIS PROTECTS AGAINST OXYGEN DAMAGE

The cytochrome b6fcomplex of oxygenic photosynthesis carries out “dark reactions” of electron transfer that link the light-driven reactions of the reaction centers, and coupled proton transfer that generates part of the electrochemical potential utilized for ATP synthesis. In contrast to the bc1 complex of the respiratory chain, with which there are many structural and functional homologies, theb6f complex contains bound pigment molecules. Along with the specifically bound chlorophyll a previously found to be bound stoichiometrically in the dimericb6f complex, it was found in the present study that β-carotene is also present in the b6fcomplex at stoichiometric levels or nearly so. Chlorophyll and carotenoid pigments were quantitatively extracted fromb6f complex purified from (i) the thermophilic cyanobacterium, Mastigocladus laminosus, (ii) spinach chloroplasts, and (iii) the green alga, Chlamydomonas reinhardtii. Visible and mass spectra showed the carotenoid to be a β-carotene of molecular weight = 536, with a stoichiometry of 1.0:1 relative to cytochrome f in the highly activeM. laminosus complex but somewhat lower stoichiometries, 0.77 and 0.55, in the b6f complex obtained from spinach chloroplasts and C. reinhardtii. A photoprotective function for the β-carotene was inferred from the findings that the rate of photobleaching of the chlorophyll a bound in the complex was found to vary inversely with β-carotene content and to decrease markedly in the presence of ambient N2 instead of air. The presence of β-carotene in the b6fcomplex, and not in the related bc1 complexes of the mitochondrial respiratory chain and photosynthetic bacteria, suggests that an additional function is to protect the protein complexes in oxygenic photosynthetic membranes against toxic effects of intramembrane singlet O2.

Structure–Function of the Cytochrome b6f Complex†

The structure and function of the cytochrome b6 f complex is considered in the context of recent crystal structures of the complex as an eight subunit, 220 kDa symmetric dimeric complex obtained from the thermophilic cyanobacterium, Mastigocladus laminosus, and the green alga, Chlamydomonas reinhardtii. A major problem confronted in crystallization of the cyanobacterial complex, proteolysis of three of the subunits, is discussed along with initial efforts to identify the protease. The evolution of these cytochrome complexes is illustrated by conservation of the hydrophobic heme‐binding transmembrane domain of the cyt b polypeptide between b6 f and bc1 complexes, and the rubredoxin‐like membrane proximal domain of the Rieske [2Fe‐2S] protein. Pathways of coupled electron and proton transfer are discussed in the framework of a modified Q cycle, in which the heme cn, not found in the bc1 complex, but electronically tightly coupled to the heme bn of the b6 f complex, is included. Crystal structures of the cyanobacterial complex with the quinone analogue inhibitors, NQNO or tridecyl‐stigmatellin, show the latter to be ligands of heme cn, implicating heme cn as an n‐side plastoquinone reductase. Existing questions include (a) the details of the shuttle of: (i) the [2Fe‐2S] protein between the membrane‐bound PQH2 electron/H+ donor and the cytochrome f acceptor to complete the p‐side electron transfer circuit; (ii) PQ/PQH2 between n‐ and p‐sides of the complex across the intermonomer quinone exchange cavity, through the narrow portal connecting the cavity with the p‐side [2Fe‐2S] niche; (b) the role of the n‐side of the b6 f complex and heme cn in regulation of the relative rates of noncyclic and cyclic electron transfer. The likely presence of cyclic electron transport in the b6 f complex, and of heme cn in the firmicute bc complex suggests the concept that hemes bn‐cn define a branch point in bc complexes that can support electron transport pathways that differ in detail from the Q cycle supported by the bc1 complex.

Evidence for a hetero-oligomeric structure of the chloroplast cytochrome b-559

A second nonhomologous polypeptide in the thylakoid membrane cytochrome b‐559 has been suggested by the finding of a smaller reading frame just slightly downstream from that corresponding to the 9 kDa cytochrome polypeptide that is dominant on a Coomassie‐stained gel. This reading frame encoded a 39‐residue polypeptide that was similar in having a central hydrophobic domain of 25–26 residues and a single His residue at the same position in the hydrophobic domain. The smallest polypeptide seen on SDS gels of the cytochrome was isolated by high‐performance liquid chromatography (HPLC). The NH2‐terminal sequence matched that of the downstream gene. The stoichiometry of the 2 gene products separated by HPLC was approx. 1:1, based on the molecular masses of 9.16 and 4.27 kDa calculated from the nucleotide sequence. It is concluded that the cytochrome contains both the 9.16 kDa (α) and 4.27 kDa (β) polypeptides. These data, the single His residue on each polypeptide, and the previous finding of a bis‐histidine coordination, imply that the unit heme binding structure of the cytochrome is a heme cross‐linked dimer. If the cytochrome contains a single heme, the dimer structure would be (αβ). If there are 2 hemes/cytochrome, the more likely structure would be (αβ)2, a tetramer consisting of 2 heme cross‐linked hetero‐dimers.

Post-translational Modifications of Integral Membrane Proteins Resolved by Top-down Fourier Transform Mass Spectrometry with Collisionally Activated Dissociation

Integral membrane proteins remain a challenge to proteomics because they contain domains with physicochemical properties poorly suited to todays bottom-up protocols. These transmembrane regions may potentially contain post-translational modifications of functional significance, and thus development of protocols for improved coverage in these domains is important. One way to achieve this goal is by using top-down mass spectrometry whereby the intact protein is subjected to mass spectrometry and dissociation. Here we describe top-down high resolution Fourier transform mass spectrometry with collisionally activated dissociation to study post-translationally modified integral membrane proteins with polyhelix bundle and transmembrane porin motifs and molecular masses up to 35 kDa. On-line LC-MS analysis of the bacteriorhodopsin holoprotein yielded b- and y-ions that covered the full sequence of the protein and cleaved 79 of 247 peptide bonds (32%). The experiment proved that the mature sequence consists of residues 14–261, confirming N-terminal propeptide cleavage and conversion of N-terminal Gln-14 to pyrrolidone carboxylic acid (−17.02 Da) and C-terminal removal of Asp-262. Collisionally activated dissociation fragments localized the N6-(retinylidene) modification (266.20 Da) between residues 225–248 at Lys-229, the sole available amine in this stretch. Off-line nanospray of all eight subunits of the cytochrome b6f complex from the cyanobacterium Nostoc PCC 7120 defined various post-translational modifications, including covalently attached c-hemes (615.17 Da) on cytochromes f and b. Analysis of murine mitochondrial voltage-dependent anion channel established the amenability of the transmembrane β-barrel to top-down MS and localized a modification site of the inhibitor Ro 68-3400 at Cys-232. Where neutral loss of the modification is a factor, only product ions that carry the modification should be used to assign its position. Although bond cleavage in some transmembrane α-helical domains was efficient, other regions were refractory such that their primary structure could only be inferred from the coincidence of genomic translation with precursor and product ions that spanned them.