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Study of the High-Potential Iron Sulfur Protein in Halorhodospira Halophila Confirms That It Is Distinct from Cytochrome C As Electron Carrier

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Study of the High-Potential Iron Sulfur Protein in Halorhodospira Halophila Confirms That It Is Distinct from Cytochrome C As Electron Carrier Study of the high-potential iron sulfur protein in Halorhodospira halophila confirms that it is distinct from cytochrome c as electron carrier Cle´ ment Lieutaud*, Jean Alric†, Marielle Bauzan‡, Wolfgang Nitschke*, and Barbara Schoepp-Cothenet*§ *Laboratoire de Bioe´nerge´tique et Inge´nierie des Prote´ines, Unite´Propre de Recherche 9036, ‡Institut de Biologie Structurale et Microbiologie, Centre National de la Recherche Scientifique, 13402 Marseille Cedex 20, France; and †Laboratoire de Ge´ne´ tique et Biophysique des Plantes, Unite´Mixte de Recherche 163, Commissariat a`l’Energie Atomique-Centre National de la Recherche Scientifique, Universite´e de la Me´diterrane´e-Commissariat a`l’Energie Atomique 1000, 13009 Marseille, France Edited by Helmut Beinert, University of Wisconsin, Madison, WI, and approved January 14, 2005 (received for review October 19, 2004) The role of high-potential iron sulfur protein (HiPIP) in donating participation of HiPIP instead of soluble cytochrome c is the rule electrons to the photosynthetic reaction center in the halophilic rather than the exception (26). For an exhaustive description of ␥-proteobacterium Halorhodospira halophila was studied by EPR electron donation to the RC in proteobacteria, therefore, de- and time-resolved optical spectroscopy. A tight complex between tailed understanding of the interaction of HiPIP with the RC is HiPIP and the reaction center was observed. The EPR spectrum of needed. HiPIP in this complex was drastically different from that of the Among the species containing HiPIPs, one stands out for its purified protein and provides an analytical tool for the detection apparent ‘‘weirdness,’’ i.e., Halorhodospira (H.) halophila. This and characterization of the complexed form in samples ranging halophilic ␥-proteobacterium has long been known to contain from whole cells to partially purified protein. The bound HiPIP was two iso-HiPIP proteins. Both differ from the bulk of HiPIPs by identified as iso-HiPIP II. Its Em value at pH 7 in the form bound to their low redox potentials (ϩ50 mV and ϩ110 mV) (27) com- .the reaction center was Ϸ100 mV higher (؉140 ؎ 20 mV) than that pared with the range of ϩ260 mV to ϩ450 mV typically found of the purified protein. EPR on oriented samples showed HiPIP II to These low redox potentials apparently argue against an impli- be bound in a well defined geometry, indicating the presence of cation of these two HiPIPs in photosynthetic electron transport. specific protein–protein interactions at the docking site. At mod- In addition, in vivo flash-induced absorbance changes have been erately reducing conditions, the bound HiPIP II donates electrons to interpreted to suggest the implication of a cytochrome rather the cytochrome subunit bound to the reaction center with a than a HiPIP in photosynthesis in this organism (26). In this half-time of <11 ␮s. This donation reaction was analyzed by using work, we have characterized the interaction of HiPIP and the RC Marcus’s outer-sphere electron-transfer theory and compared with in H. halophila in detail. It turns out that this reportedly strange those observed in other HiPIP-containing purple bacteria. The species allows profound insights into the functional character- results indicate substantial differences between the HiPIP- and the istics of HiPIP and its redox partner. cytochrome c2-mediated re-reduction of the reaction center. Experimental Procedures electron transfer ͉ photosynthesis ͉ electron paramagnetic Purification of HiPIPs. Cultures of H. halophila were grown pho- resonance ͉ redox potential ͉ reaction center tosynthetically and anaerobically at 30°C in ATCC 1448 medium in a 20-liter fermenter. igh-potential iron sulfur proteins (HiPIPs) are soluble Cells were suspended in 50 mM 3-[morpholino]propanesul- Helectron carriers containing a single cubane [Fe4S4] cluster fonic acid (Mops) at pH 7 (buffer A) and broken by passing [for reviews, see refs. 1 and 2] found in both photosynthetic (3–6) through a French press. Unbroken cells were eliminated by and nonphotosynthetic (7, 8) proteobacteria, as well as in centrifugation at low speed, and a subsequent ultracentrifuga- members of the so-called FBC group (flexibacter, bacteroides, tion separated the ‘‘total soluble fraction’’ in the supernatant cytophaga group) (9). Despite three decades of detailed bio- from the ‘‘membrane-fragments fraction’’ in the pellet. This total physical and biochemical characterization, the role of HiPIPs as soluble fraction was dialyzed to remove the remaining salt of the physiological electron donors to the reaction center (RC) in growth medium and used to purify the so-called ‘‘soluble HiPIP photosynthesis and to oxidase in respiration was not demon- I and II.’’ The membrane-fragment fraction recovered after the strated until 1995 (10–12). ultracentrifugation step was resuspended in buffer A. This HiPIPs are commonly regarded as exotic substitutes for the fraction contains almost exclusively closed vesicles that can ‘‘normal’’ soluble carrier cytochrome c. This notion is based on retain the so-called ‘‘membrane-bound HiPIP.’’ The membrane the presence of soluble cytochromes in mitochondrial respira- fragments were twice sonicated and ultracentrifuged. The su- tion, in cyanobacterial photosynthesis, and in a variety of other pernatants from both centrifugations were pooled and used to prokaryotic energy conserving systems. Moreover, the usual purify the membrane-bound HiPIP. Both soluble and mem- ‘‘workhorses’’ in the study of purple bacterial photosynthesis, brane-attached HiPIP were purified following a protocol derived Rhodobacter sphaeroides, Rhodobacter capsulatus, and Blasto- from Meyer (28). The sample was adsorbed onto a DEAE-52 chloris viridis, do not contain HiPIP. Instead, cytochrome c2, column equilibrated with buffer A. The column was eluted with together with its various iso-forms, functions in their bioener- a step-gradient from 0–0.6 M NaCl. The photoactive yellow getic chains. Several decades of studies on these species have protein was eluted at 0.12 M NaCl, the major part of the c551 at resulted in an understanding of the structural and functional 0.14, a new cytochrome c at 0.17–0.2 M (the characterization of details of electron donation from cytochrome c2 to the photo- synthetic RC (see, for example, refs. 13–19). In contrast, the interaction between HiPIP and RC is still poorly understood. This paper was submitted directly (Track II) to the PNAS office. Except for the case of Rubrivivax gelatinosus (20–23), only scarce Abbreviations: HiPIP, high-potential iron sulfur protein; RC, reaction center. data are available (24, 25). Surveys of photosynthetic electron §To whom correspondence should be addressed. E-mail: [email protected]. transfer among proteobacterial species, however, show that the © 2005 by The National Academy of Sciences of the USA 3260–3265 ͉ PNAS ͉ March 1, 2005 ͉ vol. 102 ͉ no. 9 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0407768102 Downloaded by guest on October 2, 2021 which will be presented elsewhere), HiPIP I at 0.24 M, and HiPIP II together which some cytochrome cЈ at 0.28 M. Fractions containing HiPIP I or II were dialyzed, each charged onto a second DEAE-52 column and eluted with a step-gradient from 0.2–0.24 M NaCl for HiPIP I and 0.24–0.28 M NaCl for HiPIP II. Fractions containing HiPIP I or II were dialyzed, charged onto Sephadex G-100 size-exclusion columns, and eluted with buffer A, 100 mM NaCl. Preparation of Oriented Samples. Oriented membrane multilayers were obtained by partial dehydration of the membrane frag- ments on Mylar as described by Rutherford and Se´tif (29). Ascorbate-reduced and ferricyanide-oxidized oriented samples were prepared as described by Lieutaud et al. (22). Spectroscopic Methods. EPR spectra were recorded on a Bruker (Karlsruhe, Germany) ESP 300 X-band spectrometer fitted with an Oxford Instruments (Eynsham, England) liquid helium cry- Fig. 1. EPR spectra recorded on illuminated whole cells (trace a) and on ostat and temperature control system. Illumination was per- chemically oxidized purified HiPIP I (trace b) and HiPIP II (trace c) from H. formed on whole cells resuspended in growth medium, on halophila. Instrument settings: microwave frequency, 9.44 GHz; temperature, membrane fragments diluted in buffer A, 130 g͞liter NaCl (pH 15 K; microwave power, 6.3 mW; modulation 1 mT. 7) or on membrane multilayers. During illumination, the EPR ͞ tube was kept in a water ice bath to minimize heating of the purified HiPIPs and the paramagnetic species photooxidized sample. in vivo. Optical spectra were recorded on a Cary (Victoria, Australia) 5E spectrophotometer. H. halophila Contains a HiPIP Firmly Bound to the Membrane with a Spectrum Similar to That Detected in Vivo. EPR spectra of chemi- Redox Titrations. Redox titrations were performed on membrane cally oxidized membrane fragments from H. halophila (Fig. 2, samples (after washing in 5 mM ferricyanide͞5 mM EDTA) or trace a) feature signals similar to those of illuminated whole cells. purified proteins at 15°C as described by Dutton (30) in buffer Because HiPIPs are periplasmic proteins, their copurification A with or without 130 g͞liter NaCl, in the presence of the with the membrane fraction is a priori unexpected, but compa- following redox mediators at 100 ␮M for EPR or 15 ␮M for rable results have been obtained with Rubrivivax gelatinosus (22). optical titrations: 1,4 p-benzoquinone; 2,5-dimethyl-p- Trapping of soluble HiPIPs in chromatophores might explain benzoquinone; 2-hydroxy 1,2-naphthoquinone; 1,4-naphthoqui- this finding. In the case of Rubrivivax gelatinosus, however, the none; dimethyl-1,4-naphthoquinone; 2,5-dihydroxy-p-benzoqui- absence of invaginations in the cytoplasmic membrane renders none; dihydroxy-1,4-naphthoquinone; and anthraquinone-2- trapping highly unlikely and indicates genuine physical associa- sulfonate. Ferricyanide was present at 10 ␮M. Reductive tion between HiPIP and its membrane-bound redox partners. titrations were carried out by using sodium dithionite, and oxidative titrations were carried out by using potassium hexa- Salt treatment was sufficient to dissociate the HiPIP from the chloroiridate (IV).
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