Characterization and Crystallization of the Lumen Side Domain of the Chloroplast Rieske Iron-Sulfur Protein

Abstract

A soluble, 139-residue COOH-terminal polypeptide fragment of the Rieske iron-sulfur protein of the cytochrome b 6 f complex from spinach chloroplasts was obtained by limited proteolysis of the complex and a twostep chromatography purification protocol. The purified Rieske iron-sulfur protein fragment was characterized by: (i) a single NH 2 -terminal sequence, NH 2 -Phe-Val-Pro-Pro-Gly-Gly, starting with residue 41 of the intact Rieske protein; (ii) a single molecular weight species determined by mass spectrometry with a molecular weight of 14,620 ؎ 2 without the [2Fe-2S] cluster; (iii) an optical absorbance spectrum with redox-and pHdependent maxima and minima; and (iv) a reduced-oxidized optical difference spectrum characterized by ⌬⑀ mM ‫؍‬ 3.8 mM ؊1 cm ؊1 for ⌬A at 394 versus 409 nm, which was used to determine the midpoint oxidation-reduction potential, which is ؉359 ؎ 7 mV at 25°C from pH 5.5-6.5, and ؉319 ؎ 2 mV at pH 7, with an apparent pK ox ‫؍‬ 6. The cytochrome b 6 f complex is one of three integral membrane protein complexes in the oxygenic photosynthetic membrane that participate in electron transport, H ϩ translocation, and generation of the trans-membrane proton electrochemical potential. It occupies a central position with respect to the other two integral protein systems, the photosystem II and I reaction center complexes. The Rieske high potential [2Fe-2S] iron-sulfur protein is one of four "large" polypeptides (M r ϭ 17,000 -32,000) observed in denaturing gels of the cytochrome b 6 f (plastoquinol-plastocyanin oxidoreductase) complex and one of three that contain a redox prosthetic group. In order of descending size, these four polypeptides are: cytochrome f, cytochrome b 6 , the nuclear-encoded Rieske [2Fe-2S] protein, and subunit IV, with molecular weights in the complex of spinach chloroplasts of 32,038, 24,166, 19,116, and 17,445, The first crystal structure for an active domain of the cyt b 6 f or bc 1 complexes was the 252-residue soluble lumen side fragment of the 285 residue cyt f, whose structure has been determined to a resolution of 1.96 Å (4, 5). The cytochrome was found to consist of two domains made mostly of ␤ sheet in an elongate (approximately 75 ϫ 35 ϫ 25 Å) geometry. The covalently bound heme lies within the larger domain of the elongate structure near the interface between the large and small domains, its heme iron atom 45 Å from the Arg 250 near the COOH terminus that is connected to the single trans-membrane ␣-helix. The elongate structure of cytochrome f raises questions that are defined in a structural context about the pathway of electron transfer from the membrane-bound hydrogen donor, plastoquinol, to the Rieske protein, and from the Rieske protein to cytochrome f, whose heme appears to be far from the membrane surface. In addition, the existence of a buried ordered linear H 2 O chain, a seeming proton wire with virtually all H-bond donors and acceptors conserved in evolution, which extends 11 Å from the N␦1 nitrogen of His 25 toward Lys 66 of the prominent basic su...

Electron Transfer from the Rieske Iron-Sulfur Protein (ISP) to Cytochrome f in Vitro

“…The slope of the curve in the pH-dependent region from pH 6 to 8.5 is Ϫ40 mV/pH, in agreement with the approximately Ϫ30 mV/pH obtained from optical titration in the visible spectrum (34), but smaller than the Ϫ80 mV/pH obtained for isolated bf complexes by EPR titration of the midpoint potential (35). Titration of the pH dependence of the E m of the ISP from cytochrome bc complexes, using the weak 500-nm band of the circular dichroism spectrum, yielded slopes of about Ϫ120 mV/pH at pH Ͼ 8 and Ϫ60 mV/pH below pH 8 (36,37).…”

“…4B, above), or from spinach chloroplasts determined by room temperature optical difference spectra (34), is significantly more acidic than that (7.6 -7.9) determined for the Rieske protein or a similar ISP from the bc complex in mitochondria and bacterial photosynthesis (Ͼ7.0). On the one hand, the difference of 1-1.5 pH units between the pK ox value of the chloroplast (ambient pH ϳ 6) (40) and mitochondrial/ bacterial (ambient pH ϳ 7.0) ISP agrees with the pH difference between the ambient environments in the two membrane systems, and the requirement that the ISP be able to cycle be- tween protonated and deprotonated states.…”

“…Some difference in structure between the native Rieske protein and the ISP, affecting the location relative to the [2Fe-2S] cluster of charged residues or dipole elements, is indicated by the limiting E m of the ISP at acid pH being 50 mV more positive than that of native ISP (34).…”

“…pH Titration of the Reaction of ISP and Cytochrome f-The E m of cytochrome f is pH-independent between pH 4 and 8 (33), whereas the 139-residue ISP isolated from spinach chloroplasts has a pH-dependent E m , ⌬E m (pH) ϭ Ϫ30 mV/pH (34). A larger pH dependence of the E m , ⌬E m (pH) ϭ Ϫ60 mV/pH was obtained for the ISP [2Fe-2S] cluster in isolated cytochrome b 6 f complexes from spinach (35).…”

Functional Insensitivity of the Cytochrome b6f Complex to Structure Changes in the Hinge Region of the Rieske Iron-Sulfur Protein

“…The spinach Phe 41 site (Phe 42 in Synechococcus PCC 7002), where soluble ISP was proteolyzed from the complex by thermolysin (52), is used as the start site of the linker region in the b 6 f complex. Prediction of the stop site is based on the structure of the soluble domain of spinach ISP using the start of the first ␤-strand (9).…”