Observation of deuterium-labeled diacetyldeuteroporphyrin incorporated in cyanoferrimyoglobin by deuterium nuclear magnetic resonance.

Sperm whale apomyoglobin was reconstituted with selectively deuterated D₆-2,4-diacetyldeuterohemin in which the ²H label was confined to the methyl groups of the acetyl moieties. A single resonance was observed in ²H NMR of the cyanoferrimyoglobin derivative, with a chemical shift 0.80 ppm downfield of external D₁₂-TMS at pH 6.7. The corresponding chemical shift of D₆-2,4-diacetyldeuterohemin-OMe as the cyanide complex in pyridine-water was 0.96 ppm downfield of external D₁₂-TMS. The prominent HOD peak was well separated at 4.4 ppm downfield. The line width of the porphyrin ²H resonances in both the protein and free solvent environments yields evidence of considerable rotational freedom of the -CD3 groups about their axes.     

Periplasmic electron carriers and photo-induced electron transfer in the photosynthetic bacterium Ectothiorhodospira sp.

A detailed analysis of the periplasmic electron carriers of the photosynthetic bacterium Ectothiorhodospira sp. has been performed. Two low mid-point redox potential electron carriers, cytochrome c′ and cytochrome c, are detected. A high potential iron–sulfur protein is the only high mid-point redox potential electron transfer component present in the periplasm. Analysis of light-induced absorption changes shows that this high potential iron–sulfur protein acts in vivo as efficient electron donor to the photo-oxidized high potential heme of the Ectothiorhodospira sp. reaction center. This revised version was published online in June 2006 with corrections to the Cover Date.     

Energy conservation by Rhodothermus marinus respiratory complex I

A sodium ion efflux, together with a proton influx and an inside-positive ΔΨ, was observed during NADH-respiration by Rhodothermus marinus membrane vesicles. Proton translocation was monitored by fluorescence spectroscopy and sodium ion transport by ²³Na-NMR spectroscopy. Specific inhibitors of complex I (rotenone) and of the dioxygen reductase (KCN) inhibited the proton and the sodium ion transport, but the KCN effect was totally reverted by the addition of menaquinone analogues, indicating that both transports were catalyzed by complex I. We concluded that the coupling ion of the system is the proton and that neither the catalytic reaction nor the establishment of the delta-pH are dependent on sodium, but the presence of sodium increases proton transport. Moreover, studies of NADH oxidation at different sodium concentrations and of proton and sodium transport activities allowed us to propose a model for the mechanism of complex I in which the presence of two different energy coupling sites is suggested.     

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.     

Characterization of perdeuterated high-potential iron-sulfur protein with high-resolution X-ray crystallography

Perdeuteration in neutron crystallography is an effective method for determining the positions of hydrogen atoms in proteins. However, there is shortage of evidence that the high-resolution details of perdeuterated proteins are consistent with those of the nondeuterated proteins. In this study, we determined the X-ray structure of perdeuterated high-potential iron-sulfur protein (HiPIP) at a high resolution of 0.85 å resolution. The comparison of the nondeuterated and perdeuterated structures of HiPIP revealed slight differences between the two structures. The spectroscopic and spectroelectrochemical studies also showed that perdeuterated HiPIP has approximately the same characteristics as nondeuterated HiPIP. These results further emphasize the suitability of using perdeuterated proteins in the high-resolution neutron crystallography.     

Are unit charges always negligible?

 The electrostatic contribution to the reduction potentials due to the unit charges of ionizable residues largely explains the span in redox potentials in a series of high-potential Fe₄S₄ iron-sulfur proteins and mutants. This appears to be a lucky case in which other contributions (in general) larger than that due to unit charges cancel out. Received, accepted: 26 November 1996     

Exchange integral for a variety of tetranuclear ferredoxins

The temperature dependence of EPR spectra of oxidized [4Fe-4S1]^{(?1, ?2)} ferredoxins (previously designated HiPIP) and a reduced [4Fe-4S1]^(?2,?3) ferredoxin have been analyzed so as to determine the energy of a low-lying excited electronic state. The values obtained were: Center S-3 from beef heart, 44 cm^?1; Center S-3 from mung bean, 53 cm^?1; the [4Fe-4S1]^(?1,?2) ferredoxin from Thermus thermophilus, 78 cm^?1; Center N-2 of NADH ubiquinone reductase, 83 cm^?1. Increasing axial distortion in the EPR spectra of the [4Fe-4S1]^(?1,?2) ferredoxins was associated with higher energy differences. Center N-2, a [4Fe-4S1]^(?2,?3) iron-sulfur cluster does not fit this relationship.     

Discovery and characterization of electron transfer proteins in the photosynthetic bacteria

Research on photosynthetic electron transfer closely parallels that of other electron transfer pathways and in many cases they overlap. Thus, the first bacterial cytochrome to be characterized, called cytochrome c ₂, is commonly found in non-sulfur purple photosynthetic bacteria and is a close homolog of mitochondrial cytochrome c. The cytochrome bc ₁ complex is an integral part of photosynthetic electron transfer yet, like cytochrome c ₂, was first recognized as a respiratory component. Cytochromes c ₂ mediate electron transfer between the cytochrome bc ₁ complex and photosynthetic reaction centers and cytochrome a-type oxidases. Not all photosynthetic bacteria contain cytochrome c ₂; instead it is thought that HiPIP, auracyanin, Halorhodospira cytochrome c551, Chlorobium cytochrome c555, and cytochrome c ₈ may function in a similar manner as photosynthetic electron carriers between the cytochrome bc ₁ complex and reaction centers. More often than not, the soluble or periplasmic mediators do not interact directly with the reaction center bacteriochlorophyll, but require the presence of membrane-bound intermediates: a tetraheme cytochrome c in purple bacteria and a monoheme cytochrome c in green bacteria. Cyclic electron transfer in photosynthesis requires that the redox potential of the system be delicately poised for optimum efficiency. In fact, lack of redox poise may be one of the defects in the aerobic phototrophic bacteria. Thus, large concentrations of cytochromes c ₂ and c′ may additionally poise the redox potential of the cyclic photosystem of purple bacteria. Other cytochromes, such as flavocytochrome c (FCSD or SoxEF) and cytochrome c551 (SoxA), may feed electrons from sulfide, sulfur, and thiosulfate into the photosynthetic pathways via the same soluble carriers as are part of the cyclic system. This revised version was published online in August 2006 with corrections to the Cover Date.