High-potential Iron-sulfur Protein Research Articles

Experimental evidence for the role of buried polar groups in determining the reduction potential of metalloproteins: the S79P variant of Chromatium vinosum HiPIP.

The amide group between residues 78 and 79 of Chromatium vinosum high-potential iron-sulfur protein (HiPIP) is in close proximity to the Fe4S4 cluster of this protein and interacts via a hydrogen bond with S gamma of Cys77, one of the cluster ligands. The reduction potential of the S79P variant was 104 +/- 3 mV lower than that of the recombinant wild-type (rcWT) HiPIP (5 mM phosphate, 100 mM NaCl, pH 7, 293 K), principally due to a decrease in the enthalpic term which favors the reduction of the rcWT protein. Analysis of the variant protein by NMR spectroscopy indicated that the substitution has little effect on the structure of the HiPIP or on the electron distribution in the oxidized cluster. Potential energy calculations indicate that the difference in reduction potential between rcWT and S79P variant HiPIPs is due to the different electrostatic properties of amide 79 in these two proteins. These results suggest that the influence of amide group 79 on the reduction potential of C. vinosum HiPIP is a manifestation of a general electrostatic effect rather than a specific interaction. More generally, these results provide experimental evidence for the importance of buried polar groups in determining the reduction potentials of metalloproteins.

Amino Acid Sequences of Two High-Potential Iron–Sulfur Proteins (HiPIPs) from the Moderately Halophilic Purple Phototrophic Bacterium, Rhodospirillum salinarum

The amino acid sequences of two very different high-potential iron–sulfur protein (HiPIP) isozymes have been determined from the moderately halophilic purple phototrophic bacterium, Rhodospirillum salinarum. Iso-1 HiPIP, which is monomeric and contains 57 amino acid residues, is most similar to the Thiobacillus ferrooxidans iron-oxidizing enzyme (45% identity and a 6-residue deletion). On the other hand, iso-2 HiPIP, which is isolated as an oligomer, contains a peptide chain with 54 amino acid residues. It is the smallest reported to date and is only 31% identical to iso-1 HiPIP. A massive deletion of 17 residues is found at the N-terminus, such that only 2 residues remain prior to the first cysteine. Iso-2 HiPIP also has a 12-residue insertion and a 5-residue deletion. Prior to this study, there were only 2 absolutely conserved residues (Tyr 19 and Gly 75, Chromatium numbering) in addition to the 4 iron–sulfur cluster binding cysteine residues among the 13 HiPIPs sequenced to date. We found that Tyr 19 is absent in iso-2 HiPIP along with the entire N-terminal loop. Moreover, Gly 75 is substituted in both R. salinarum HiPIPs. These characteristics make the R. salinarum HiPIPs, and especially iso-2, the most divergent yet characterized.

The caa3 terminal oxidase of the thermohalophilic bacterium Rhodothermus marinus: a HiPIP:oxygen oxidoreductase lacking the key glutamate of the D-channel

The respiratory chain of the thermohalophilic bacterium Rhodothermus marinus contains a novel complex III and a high potential iron-sulfur protein (HiPIP) as the main electron shuttle (Pereira et al., Biochemistry 38 (1999) 1268–1275 and 1276–1283). In this paper, one of the terminal oxidases expressed in this bacterium is extensively characterised. It is a caa 3-type oxidase, isolated with four subunits (apparent molecular masses of 42, 19 and 15 kDa and a C-haem containing subunit of 35 kDa), which has haems of the A s type. This oxidase is capable of using TMPD and horse heart cytochrome c as substrates, but has a higher turnover with HiPIP, being the first example of a HiPIP:oxygen oxidoreductase. The oxidase has unusually low reduction potentials of 260 (haem C), 255 (haem A) and 180 mV (haem A 3). Subunit I of R. marinus caa 3 oxidase has an overall significant homology with the subunits I of the COX type oxidases, namely the metal binding sites and most residues considered to be functionally important for proton uptake and pumping (K- and D-channels). However, a major difference is present: the putative essential glutamate (E278 in Paraccocus denitrificans) of the D-channel is missing in the R. marinus oxidase. Homology modelling of the R. marinus oxidase shows that the phenol group of a tyrosine residue may occupy a similar spatial position as the glutamate carboxyl, in relation to the binuclear centre. Moreover, sequence comparisons reveal that several enzymes lacking that glutamate have a conserved substitution pattern in helix VI: -YSHPXV- instead of -XGHPEV-. These observations are discussed in terms of the mechanisms for proton uptake and it is suggested that, in these enzymes, tyrosine may play the role of the glutamate in the proton channel.

Investigation of the role of a surface patch in the self-association of Chromatium vinosum high potential iron-sulfur protein

The role of a flattened, relatively hydrophobic surface patch in the self-association of Chromatium vinosum HiPIP was assessed by substituting phenylalanine 48 with lysine. The reduction potential of the F48K variant was 26 mV higher than that of the wild-type (WT) recombinant (rc) HiPIP, consistent with the introduction of a positive charge close to the cluster. Nuclear magnetic resonance spectroscopy (NMR) revealed that the electronic structure of the oxidized cluster in these two proteins is very similar at 295 K. In contrast, the electron transfer self-exchange rate constant of F48K was at least 15-fold lower than that of the WT rcHiPIP, indicating that the introduction of a positive charge at position 48 diminishes self-association of the HiPIP in solution. Moreover, the substitution at position 48 abolished the fine structure in the g z region of the electron paramagnetic resonance (EPR) spectrum of oxidized C. vinosum rcHiPIP recorded in the presence of 1 M sodium chloride. These results support the hypothesis that the flattened, relatively hydrophobic patch mediates interaction between two molecules of HiPIP and that freezing-induced dimerization of the HiPIP mediated by this patch is responsible for the unusual fine structure observed in the EPR spectrum of the oxidized C. vinosum HiPIP.

Structural and dynamical properties of a partially unfolded Fe4S4 protein: role of the cofactor in protein folding.

Heteronuclear multidimensional NMR spectroscopy was used to investigate in detail the structural and dynamical properties of a partially unfolded intermediate of the reduced high-potential iron-sulfur protein (HiPIP) from Chromatium vinosum present in 4 M guanidinium chloride solution. After an extensive assignment of 15N and 1H resonances, NOE data, proton longitudinal relaxation times, and 3JHNHalpha coupling constants as well as 15N relaxation parameters (T1, T2, T1rho, and 1H-15N NOE) were obtained and used to build a structural model of the intermediate. The Fe4S4 cluster of the HiPIP plays a decisive role in determining the resulting structure, which is random in the N-terminal half of the protein and partially organized in the loops between the cysteines bound to the cluster. Consistent with the structural data, the backbone mobility is typical of folded proteins in the regions where there are elements of structure and increases with the structural indetermination.

Determination of Basicity of Core X and Terminal Y Ligands (X, Y = S and Se) of Reduced, Oxidized, and Super-Oxidized Forms of [Fe4X4(YAd)4]2− (Ad = 1-Adamantyl) in Aqueous Solutions

Abstract A series of [Fe4X4(YAd)4]2− (X, Y = S, Se; Ad = 1-adamantyl) were prepared as a model of high potential iron–sulfur proteins. Hydrolysis of the clusters was effectively depressed in aqueous poly[2-(dimethylamino)hexanamide] (PDAH) solutions due to the enbedding effect in hydrophobic environment and/or inhibition of dissociation of the terminal ligand into the aqueous media. Cyclic voltammetry of those clusters in aqueous PDAH solutions showed pH-dependent redox potentials of not only the [Fe4X4]+/2+ but also the [Fe4X4]2+/3+ (X = S and Se) couples, resulting from redox-linked protonation reactions of three oxidation states of [Fe4X4(YAd)4]n− (n = 1—3). pKavalues of the reduced, oxidized, and super-oxidized forms of [Fe4X4(YAd)4]2− were determined by computer simulation of the pH dependent redox potentials. The basicity of the core X (X = S and Se) of three oxidation states of [Fe4X4(YAd)4]n− (n = 1, 2, 3) was stronger than the terminal YAd (Y = S and Se) ligands: in the case of the mono-protonated [Fe4X4(YAd)4]3−(H+) and [Fe4X4(YAd)4]2−(H+), basicity of the terminal YAd ligand of [Fe4X4(YAd)4]2−(H+) becomes stronger than that of core X, although the core X of [Fe4X4(YAd)4]3−(H+) still showed a stronger basicity than those of terminal YAd ligands.

Interaction site for high-potential iron-sulfur protein on the tetraheme cytochrome subunit bound to the photosynthetic reaction center of Rubrivivax gelatinosus.

We have recently demonstrated, using site-directed mutagenesis, that soluble cytochromes interact with the Rubrivivax gelatinosus photosynthetic reaction center (RC) in the vicinity of the low-potential heme 1 (c-551, Em = 70 mV) of the tetraheme cytochrome subunit, the fourth heme from the special pair of bacteriochlorophyll [Osyczka, A., et al. (1998) Biochemistry 37, 11732-11744]. Although the mutations generated in that study did not show clear effects on the electron transfer from high-potential iron-sulfur protein (HiPIP), which is the major physiological electron donor to the RC in this bacterium, we report here that other site-directed mutations near the solvent-exposed edge of the same low-potential heme 1, V67K (valine-67 substituted by lysine) and E79K/E85K/E93K (glutamates-79, -85, and -93, all replaced by lysines), considerably inhibit the electron transfer from HiPIP to the RC. Thus, it is concluded that HiPIP, like soluble cytochromes, binds to the RC in the vicinity of the exposed part of the low-potential heme 1 of the cytochrome subunit, although some differences in the configurations of the HiPIP-RC and cytochrome c-RC transient complexes may be postulated.

On the role of high-potential iron–sulfur proteins and cytochromes in the respiratory chain of two facultative phototrophs

The capability of high potential iron–sulfur proteins (HiPIPs) and soluble cytochromes to shuttle electrons between the bc 1 complex and the terminal oxidase in aerobically grown cells of Rhodoferax fermentans and Rhodospirillum salinarum, two facultative phototrophs, was evaluated. In Rs. salinarum, HiPIP and a c-type cytochrome (α-band at 550 nm, E m,7=+290 mV) are both involved in the electron transfer step from the bc 1 complex to the terminal oxidase. Kinetic studies indicate that cytochrome c 550 is more efficient than HiPIP in oxidizing the bc 1 complex, and that HiPIP is a more efficient reductant of the terminal oxidase as compared to cytochrome c 550. Rs. salinarum cells contain an additional c-type cytochrome (asymmetric α-band at 556 nm, E m,7=+180 mV) which is able to reduce the terminal oxidase, but unable to oxidize the bc 1 complex. c-type cytochromes could not be isolated from Rf. fermentans, in which HiPIP, the most abundant soluble electron carrier, is reduced by the bc 1 complex (zero-order kinetics) and oxidized by the terminal oxidase (first-order kinetics), respectively. These data, taken together, indicate for the first time that HiPIPs play a significant role in bacterial respiratory electron transfer.

Membrane-bound electron transfer chain of the thermohalophilic bacterium Rhodothermus marinus: characterization of the iron-sulfur centers from the dehydrogenases and investigation of the high-potential iron-sulfur protein function by in vitro reconstitution of the respiratory chain.

Rhodothermus marinus, a thermohalophilic bacterium, has a unique electron-transfer chain, containing, besides a cbb3 and a caa3 terminal oxidases, a novel cytochrome bc complex [Pereira, M. M., Carita, J. N., and Teixeira, M. (1999) Biochemistry 38, 1268-1275]. The membrane-bound iron-sulfur centers of this bacterium were studied by electron paramagnetic resonance (EPR) spectroscopy, leading to the identification of its main electron-transfer complexes. The resonances typical for the Rieske-type centers are not detected. Clusters S1 and S3 from succinate dehydrogenase were identified; interestingly, center S3 is shown to be present in two different conformations, with g values at 2.035, 2.009, and 2.001 and at 2.025, 2.002, and 2.000. Upon addition of NADH and dithionite, EPR signals assigned to resonances characteristic of binuclear and tetranuclear clusters develop and are attributed to the iron-sulfur centers of complexes I and II. A high-potential iron-sulfur protein- (HiPIP-) type center previously detected in the membranes of this bacterium [Pereira et al. (1994) FEBS Lett. 352, 327-330] is shown to belong indeed to a canonical HiPIP. This protein was purified and extensively characterized. It is a small water-soluble protein of approximately 10 kDa, containing a single [4Fe-4S]3+/2+ cluster. The reduction potential, determined by EPR redox titrations in intact and detergent-solubilized membranes as well as by cyclic voltammetry in solution, has a pH-independent value of 260 +/- 20 mV, in the range 6-9. In vitro reconstitution of the R. marinus electron-transfer chain shows that the HiPIP plays a fundamental role in the chain, as the electron shuttle between R. marinus cytochrome bc complex and the caa3 terminal oxidase, being thus simultaneously identified a HiPIP reductase and a HiPIP oxidase.

Membrane-bound electron transfer chain of the thermohalophilic bacterium Rhodothermus marinus: a novel multihemic cytochrome bc, a new complex III.

A novel multihemic cytochrome bc complex was isolated from the membranes of Rhodothermus marinus. It is a complex with a minimum of three subunits (43, 27, and 18 kDa), containing five low-spin heme centers of the B and C types, in a 1:4 ratio. All the C-type hemes are in the same subunit (27 kDa). Three distinct redox transitions, at 235, 80, and -45 mV, were observed by visible redox titrations. The first involves one B- and one C-type hemes, and in the other two transitions one and two C-type hemes are involved, respectively. Spectroscopic data strongly suggest that the two hemes intervening in the last transition are in van der Waals contact, yielding a split Soret band. Electron paramagnetic resonance spectra of the oxidized complex show resonances of five low-spin ferric heme centers. Upon reduction with ascorbate, all these resonances vanish and a new one attributed to the last pair of hemes appears. A [3Fe-4S]1+/0 center copurifies with this complex, having a high reduction potential of +140 mV. No Rieske-type centers are detected in R. marinus and no effect is observed in the respiratory rates when the typical bc1 complex inhibitors are present, suggesting that such a complex is absent in R. marinus [Pereira et al. (1994) FEBS Lett. 352, 327-330]. The newly isolated cytochrome bc complex has quinol:cytochrome c or high-potential iron-sulfur protein (HiPIP) oxidoreductase activity, being a functional analogue of the canonical bc1 complexes; i.e., it is the complex III in R. marinus. This complex plays a central role in this bacterium's electron-transfer chain, coupling the electron transfer between the quinols reduced by the dehydrogenases and the HiPIP, the final electron donor to the terminal oxidases [Pereira, M. M., Carita, J. N., and Teixeira, M. (1999) Biochemistry 38, 1276-1283].

The respiratory chain of the halophilic anoxygenic purple bacterium Rhodospirillum sodomense.

The halophilic purple nonsulfur bacterium Rhodospirillum sodomense has been previously described as an obligate phototroph that requires yeast extract and a limited number of organic compounds for photoheterotrophic growth. In this work, we report on chemoheterotrophic growth of R. sodomense in media containing either acetate or succinate supplemented with 0.3-0.5% yeast extract. Plasma membranes isolated from cells grown aerobically in the dark contained three b-type and three c-type membrane-bound cytochromes with Em,7 of +171 +/- 10, +62 +/- 10 and -45 +/- 13 mV (561-575 nm), and +268 +/- 6, +137 +/- 10 and -43 +/- 12 mV (551-540 nm). A small amount of a soluble c-type cytochrome with a mol. mass of 15 kDa (Em, 7 >/= +150 mV) was identified. Spectroscopic and immunological methods excluded the presence of cytochrome of the c2 class and high-potential iron-sulfur proteins. Inhibitory studies indicated that only 60-70% of the respiratory activity was blocked by low concentrations of cyanide, antimycin A, and myxothiazol (10, 0.1, and 0.2 microM, respectively). These results were interpreted to show that the oxidative electron transport chain of R. sodomense is branched, leads to a quinol oxidase that is fully blocked by 1 mM cyanide and that is involved in light-dependent oxygen reduction, and leads to a cytochrome c oxidase that is inhibited by 10 microM cyanide. These features taken together suggest that R. sodomense differs from the closely related species Rhodospirillum salinarum and from other species of the genus Rhodospirillum in that it contains multiple membrane-bound cytochromes c.

Crystal Structure and Possible Dimerization of the High-Potential Iron−Sulfur Protein fromChromatiumpurpuratum,

The crystal structure of the high-potential iron-sulfur protein (HiPIP) isolated from Chromatium purpuratum is reported at 2.7 A resolution. The three HiPIP molecules in the asymmetric unit of the crystals form one and one-half dimers. Two molecules are related by a noncrystallographic symmetry rotation of approximately 175 degrees with negligible translation along the dyad axis. The third molecule in the asymmetric unit also forms a dimer with a second HiPIP molecule across the crystallographic 2-fold symmetry axis. The Fe4S4 clusters in both the crystallographic and noncrystallographic dimers are separated by approximately 13.0 A. Solution studies give mixed results regarding the oligomeric state of the C. purpuratum HiPIP. A comparison with crystal structures of HiPIPs from other species shows that HiPIP tends to associate rather nonspecifically about a conserved, relatively hydrophobic surface patch to form dimers.

Evidence against the double-arginine motif as the only determinant for protein translocation by a novel Sec-independent pathway in Escherichia coli

Proteins which are synthesized with a signal peptide containing a `double-arginine' motif may be translocated across the bacterial cytoplasmic membrane by a mechanism that is different from the known Sec and signal recognition particle pathways. The function of the double-arginine motif as a determinant for this novel pathway was studied by expressions of gene constructs coding for the high potential iron-sulfur protein (HiPIP) from Chromatium vinosum D in Escherichia coli. When the protein was produced with its original double-arginine motif-containing signal peptide, it was in part translocated into the periplasm and thereby processed, as shown by immunoblots after cell fractionation and N-terminal sequencing of purified HiPIP. Processing was not inhibited significantly by 3 mM sodium azide, indicating that translocation of HiPIP occurs by a SecA-independent pathway. Translocation of HiPIP could be altered to the SecA-dependent mode when its signal peptide was substituted by that of PelB from Erwinia carotovora. When the HiPIP double-arginine motif (S RR DAVK) was introduced into the corresponding position of the PelB signal peptide, the transport pathway remained SecA-dependent. This indicates that additional determinants are required for translocation by the Sec-independent pathway.

Evidence against the double-arginine motif as the only determinant for protein translocation by a novel Sec-independent pathway in Escherichia coli.

Proteins which are synthesized with a signal peptide containing a 'double-arginine' motif may be translocated across the bacterial cytoplasmic membrane by a mechanism that is different from the known Sec and signal recognition particle pathways. The function of the double-arginine motif as a determinant for this novel pathway was studied by expressions of gene constructs coding for the high potential iron-sulfur protein (HiPIP) from Chromatium vinosum D in Escherichia coli. When the protein was produced with its original double-arginine motif-containing signal peptide, it was in part translocated into the periplasm and thereby processed, as shown by immunoblots after cell fractionation and N-terminal sequencing of purified HiPIP. Processing was not inhibited significantly by 3 mM sodium azide, indicating that translocation of HiPIP occurs by a SecA-independent pathway. Translocation of HiPIP could be altered to the SecA-dependent mode when its signal peptide was substituted by that of PelB from Erwinia carotovora. When the HiPIP double-arginine motif (SRRDAVK) was introduced into the corresponding position of the PelB signal peptide, the transport pathway remained SecA-dependent. This indicates that additional determinants are required for translocation by the Sec-independent pathway.

Thermodynamics of reduction of Chromatium vinosum high-potential iron-sulfur protein and its histidine depleted H42Q mutant

The thermodynamic parameters for the reduction reaction of the high-potential iron-sulfur protein from Chromatium vinosum, and its H42Q mutant, have been measured by direct cyclic voltammetry using a ‘non-isothermal’ reference electrode. Histidine 42 is mainly responsible for the pH dependence of the reduction potential ( E°) with p K a values equal to 5.9 and 6.3 for the oxidized and reduced proteins, respectively. The H42Q mutant does not present this pH dependence, although an increasing E° at the lowest pH values is still present. The main contribution to the difference in free energy between the protonated and the neutral His42 forms is given by the enthalpic term ( ΔΔH° = 7.4 kmol −1), although the entropic term is not negligible.

ROLE OF HIPIP AS ELECTRON DONOR TO THE RC-BOUND CYTOCHROME IN PHOTOSYNTHETIC PURPLE BACTERIA

High-Potential Iron-Sulfur Proteins (HiPIP) are small electron carriers, present only in species of photosynthetic purple bacteria having a RC-bound cytochrome. Their participation in the photo-induced cyclic electron transfer was recently established for Rubrivivax gelatinosus, Rhodocyclus tenuis and Rhodoferax fermentans (Schoepp et al. 1995; Hochkoeppler et al. 1996a, Menin et al. 1997b). To better understand the physiological role of HiPIP, we extended our study to other selected photosynthetic bacteria. The nature of the electron carrier in the photosynthetic pathway was investigated by recording light-induced absorption changes in intact cells. In addition, EPR measurements were made in whole cells and in membrane fragments in solution or dried immobilized, then illuminated at room temperature. Our results show that HiPIP plays an important role in the reduction of the photo-oxidized RC-bound cytochrome in the following species: Ectothiorhodospira vacuolata, Chromatium vinosum, Chromatium purpuratum and Rhodopila globiformis. In Rhodopseudomonas marina, the HiPIP is not photo-oxidizible in whole cells and in dried membranes, suggesting that this electron carrier is not involved in the photosynthetic pathway. In Ectothiorhodospira halophila, the photo-oxidized RC-bound cytochrome is reduced by a high midpoint potential cytochrome c, in agreement with midpoint potential values of the two iso-HiPIPs (+ 50 mV and + 120 mV) which are too low to be consistent with their participation in the photosynthetic cyclic electron transfer.

Identification of slow motions in the reduced recombinant high-potential iron sulfur protein I (HiPIP I) from Ectothiorhodospira halophila via 15N rotating-frame NMR relaxation measurements.

Rotating-frame 15N relaxation rate (R1 rho) NMR experiments have been performed in order to study the dynamic behavior of the reduced recombinant high-potential iron-sulfur protein iso I (HiPIP I) from Ectothiorhodospira halophila, in the microsecond to ms time range. Measurements of R1 rho were performed as a function of the effective spinlock magnetic field amplitude by using both on and off-resonance radio frequency irradiation. The two data sets provided consistent results and were fit globally in order to identify possible exchange processes in an external loop of the reduced HiPIP I. The loop consists of residues 43-45 and the correlation time of the exchange process was determined to be 50 +/- 8 microseconds for the backbone nitrogen of Gln 44.

Local mobility of 15N labeled biomolecules characterized through cross-correlation rates: Applications to paramagnetic proteins.

The mobility of (15)N labeled proteins can be characterized by measuring the cross-correlation rates delta(N,NI) that govern the conversion of Zeeman order N(z) of an amide (15)N nucleus into longitudinal two-spin order 2N(z)I(z) involving the amide (15)N and (1)H nuclei. This represents an alternative to the measurement of (15)N self-relaxation rates 1/T(1) and 1/T(2) or 1/T(1rho). The rate of interconversion between N(z) and 2N(z)I(z) is due to cross-correlation between fluctuations of different interactions and is not affected by a variety of relaxation mechanisms that contribute to the self-relaxation rates 1/T(1), 1/T(2) and 1/T(1rho). Spin diffusion among protons, which affects the measurements, can be quenched by various means that are evaluated by experiments and simulations. By applying an off-resonance radio-frequency (RF) field in the vicinity of the nitrogen resonance, the spectral density function J(omega) can be determined at the frequency origin and at the nitrogen Larmor frequency. The methods are applied to the paramagnetic High-Potential Iron-Sulfur Protein iso I (HiPIP I) from E. halophila in its reduced state.

Photoinduced cyclic electron transfer in Rhodocyclus tenuis cells: participation of HiPIP or cyt c8 depending on the ambient redox potential.

We demonstrate the participation of a cytochrome c8 and a high-potential iron-sulfur protein (HiPIP) in the photoinduced electron transfer in whole cells of Rhodocyclus tenuis depending on the redox state or background continuous illumination. At high redox potentials (above 350 mV) or under a strong background illumination (5 W m-2), the cytochrome c8 acts as the physiological electron donor to the photo-oxidized high-potential hemes of the tetraheme cytochrome bound to the reaction center. For redox potentials ranging from 200 to 310 mV or under weak background illumination (1. 25 W m-2), the electron carrier is the HiPIP. The electron transfer between cyt c8 and HiPIP and the tetraheme cytochrome has half-times of 300 and 480 micros, respectively. A slow electrogenic phase of the membrane potential is linked to their rereduction. This phase is sensitive to a specific inhibitor of the cyt bc1 complex, indicating involvement of cyt c8 and HiPIP in the photoinduced cyclic electron transfer at these two redox conditions.

The electron transport system of the halophilic purple nonsulfur bacterium Rhodospirillum salinarum. 1. A functional and thermodynamic analysis of the respiratory chain in aerobically and photosynthetically grown cells.

Plasma membranes isolated from cells of the halophilic purple nonsulfur bacterium Rhodospirillum salinarum grown in light or in the dark were examined. Membranes isolated from cells grown aerobically in the dark contained three b-type and two c-type membrane-bound cytochromes with Em,7 of +180, +72 and -5 mV (561-575 nm), and +244 and +27 mV (551-540 nm), respectively. Conversely, membranes isolated from cells grown anaerobically in the light contained two b-type and five c-type haems with Em,7 of +60 and -45 mV and +290, +250, +135, -20 and -105 mV, respectively. In addition to haems of the b- and c-type, two haems of the a-type (Em,7 of +325 and +175 mV) were present only in cells grown in the dark. Four soluble cytochromes of the c type, but not cytochrome c2, along with two high-potential iron-sulfur proteins (HiPIP iso-1 and iso-2) were also identified in cells grown aerobically. Inhibitory studies showed that 85-90% of the respiratory activity was blocked by very low concentrations of cyanide, antimycin A and myxothiazol (50, 0.1 and 0.2 mM, respectively). These results taken together were interpreted to show that the oxidative electron transport chain of Rsp. salinarum is linear, leading to a membrane-bound oxidase of the aa3 type in cells grown in the dark, while no significant cytochrome oxidase activity is catalyzed by photosynthetic membranes. These features suggest that this halophilic species is unique among the genus Rhodospirillum and that it also differs from other facultative phototrophs (e.g., Rhodobacter species) in that it does not contain either cytochrome c2 or a branched respiratory chain.