Structure-Function, Stability, and Chemical Modification of the Cyanobacterial Cytochrome b6f Complex from Nostoc sp. PCC 7120

Abstract

The crystal structure of the cyanobacterial cytochrome b(6)f complex has previously been solved to 3.0-A resolution using the thermophilic Mastigocladus laminosus whose genome has not been sequenced. Several unicellular cyanobacteria, whose genomes have been sequenced and are tractable for mutagenesis, do not yield b(6)f complex in an intact dimeric state with significant electron transport activity. The genome of Nostoc sp. PCC 7120 has been sequenced and is closer phylogenetically to M. laminosus than are unicellular cyanobacteria. The amino acid sequences of the large core subunits and four small peripheral subunits of Nostoc are 88 and 80% identical to those in the M. laminosus b(6)f complex. Purified b(6)f complex from Nostoc has a stable dimeric structure, eight subunits with masses similar to those of M. laminosus, and comparable electron transport activity. The crystal structure of the native b(6)f complex, determined to a resolution of 3.0A (PDB id: 2ZT9), is almost identical to that of M. laminosus. Two unique aspects of the Nostoc complex are: (i) a dominant conformation of heme b(p) that is rotated 180 degrees about the alpha- and gamma-meso carbon axis relative to the orientation in the M. laminosus complex and (ii) acetylation of the Rieske iron-sulfur protein (PetC) at the N terminus, a post-translational modification unprecedented in cyanobacterial membrane and electron transport proteins, and in polypeptides of cytochrome bc complexes from any source. The high spin electronic character of the unique heme c(n) is similar to that previously found in the b(6)f complex from other sources.

FIGURE 1.

Density centrifugation of Nostoc cytochrome b₆f fractions collected from the hydrophobic chromatography column. The two cytochrome b₆f containing fractions, which eluted in the first fractions from the column (left), and in the late fractions (right), were concentrated, loaded onto 10-ml 8–35% sucrose gradient in the TNE buffer supplemented with 1 mm UDM, and centrifuged at 200,000 × g (16 h) in an SW-41 rotor (Beckman-Coulter). Cytochrome b₆f monomer and dimer bands are indicated by arrows.

FIGURE 2.

Partial N-terminal acetylation of the Rieske iron-sulfur protein. LC-MS of the cytochrome b₆f complex from Nostoc reveals two populations of Rieske ISP. A, electrospray-ionization mass spectrum of Rieske ISP after zero-charge deconvolution reveals sub-populations at 19,064.2 and 19,106.6 Da, consistent with partial acetylation (+42 Da). Specific ion chromatograms for each species are shown in the inset, revealing that the putative acetylated form is more highly retained, consistent with it being more hydrophobic due to having one less charge. B, analysis of an N-terminal tryptic peptide of Rieske ISP by tandem mass spectrometry using collisionally activated dissociation. The collisionally activated dissociation spectrum shown was annotated with respect to the important b-ion series (b₁–b₅) that are all consistent with N-terminal acetylation. The table was generated by the Mascot algorithm (Matrix Sciences), which picked the N-terminally acetylated N-terminal tryptic peptide of Nostoc Rieske ISP out of the complete protein data base (MSDB at Matrix Sciences on 11/11/08; search run in “no enzyme” mode) as the best match to the experimental dataset with a score of 40 (see Ref. 18). The matched ions are shown in boldface and localize the delta 42-Da modification to the N-terminal amino acid residue.

FIGURE 3.

Ribbon diagram of the symmetric dimeric structure of the cytochrome b₆f complex from Nostoc sp. PCC 7120 determined from the 3.0-Å structure. The color code for the p-side of complex are: red, cyt f (petA); yellow, Rieske ISP (petA); blue, cytb (petB); purple, subunit IV; and green, petG, -L, -M, and -N. p-side heme of cyt f and [2Fe-2S] cluster are shown, along with two trans-membrane b-hemes, n-side heme c_n represented as stick diagrams. The bound chlorophyll a and β-carotene are shown in stick format, in dark green and in orange, respectively. The inset shows bipyramidal crystals of the b₆f complex from Nostoc obtained using the hanging drop, vapor-diffusion method described under “Experimental Procedures” (bar size, 100 μm).

FIGURE 4.

X-band EPR spectra of cytochrome b₆f complex from Nostoc for parallel and perpendicular orientations of the microwave magnetic field. Purified b₆f complex was diluted to a concentration of 0.105 mm in TNE buffer supplemented with 1 mm UDM and 300 mm sucrose. Experimental conditions: microwave frequency, 9.65 GHz (B₁ ⊥ B) and 9.37 GHz (B₁ ∥ B); microwave power, 2 milliwatts (B₁ ⊥ B) and 0.2 milliwatt (B₁ ∥ B).