We report on room temperature electron transfer in the reaction center (RC) complex purified from Rhodobacter sphaeroides. The protein was embedded in trehalose-water systems of different trehalose/water ratios. This enabled us to get new insights on the relationship between RC conformational dynamics and long-range electron transfer. In particular, we measured the kinetics of electron transfer from the primary reduced quinone acceptor (Q(A)(-)) to the primary photo oxidized donor (P(+)), by time-resolved absorption spectroscopy, as a function of the matrix composition. The composition was evaluated either by weighing (liquid samples) or by near infrared spectroscopy (highly viscous or solid glasses). Deconvolution of the observed, nonexponential kinetics required a continuous spectrum of rate constants. The average rate constant ( = 8.7 s(-1) in a 28% (w/w) trehalose solution) increases smoothly by increasing the trehalose/water ratio. In solid glasses, at trehalose/water ratios > or = 97%, an abrupt increase is observed ( = 26.6 s(-1) in the driest solid sample). A dramatic broadening of the rate distribution function parallels the above sudden increase. Both effects fully revert upon rehydration of the glass. We compared the kinetics observed at room temperature in extensively dried water-trehalose matrices with the ones measured in glycerol-water mixtures at cryogenic temperatures and conclude that, in solid trehalose-water glasses, the thermal fluctuations among conformational substates are inhibited. This was inferred from the large broadening of the rate constant distribution for electron transfer obtained in solid glasses, which was due to the free energy distribution barriers having become quasi static. Accordingly, the RC relaxation from dark-adapted to light-adapted conformation, which follows primary charge separation at room temperature, is progressively hindered over the time scale of P(+)Q(A)(-) charge recombination, upon decreasing the water content. In solid trehalose-water glasses the electron transfer process resulted much more affected than in RC dried in the absence of sugar. This indicated a larger hindering of the internal dynamics in trehalose-coated RC, notwithstanding the larger amount of residual water present in comparison with samples dried in the absence of sugar.

This study shows that the product of the hoxZ gene of Alcaligenes eutrophus HI6 is a 6-type cytochrome (cytochrome bJ, which is essential for anchoring the membrane-bound hydrogenase (MBH) complex to the periplasmic side of the membrane and for H,-coupled respiration. The hoxZ product is not required for MBH translocation and H,-dependent reduction of the redox dye, 2,3,5-triphenyl-2-tetrazolium chloride. The lack of cytochrome b, does not affect the electron-transport activities linked to oxidation of succinate and NADH, although it enhances the electron-flow rate through the cytochrome-c oxidase pathway in hoxZA membranes. We show that the hoxZ product is a dihaem cytochrome b (haems with of + 10 mV and + 166 mV) involved in H,-dependent electron transfer. We conclude that cytochrome b, of the A. eutrophus MBH complex is the link necessary for transfer of electrons from H, to the ubiquinone pool and that it is required for attachment of MBH to the membrane.Keywords: cytochrome b subunit; hydrogen respiration ; hoxZ gene; membrane-bound hydrogenase; Alcaligenes eutrophus.Alcaligenes eutrophus H16, a gram-negative respiration-dependent bacterium, belongs to the group of facultative lithoautotrophs that can use hydrogen as sole energy source [l]. Oxidation of hydrogen is mediated by two [NiFeI-containing hydrogenases : a cytoplasmic, heterotetrameric NAD-reducing enzyme (SH), which consists of four subunits encoded by the genes hoxF, hoxU, hoxH and hoxY [ 2 ] ; and a membrane-bound hydrogenase (MBH), which is composed of a small and a large subunit, encoded by the genes hoxK and hoxC, respectively [3]. The structural genes for SH and MBH are arranged in two separate operons tightly clustered with sets of accessory genes that are required for the formation of enzymatically active hydrogenase. The accessory-gene products participate in a series of complex post-translational events involving metal-center assembly, C-terminal proteolytic processing, oligomerization and, in the case of the MBH, translocation [4 -61.Crystal-structure analysis of the periplasmic [NiFe] hydrogenase of Desulfovibrio gigas has shown that the binuclear metal center is deep inside the protein and that the mature large subunit is devoid of a C-terminal extension [7]. Evidence for the presence of specific proteases that remove at least 15 C-terminal residues has been documented in a variety of phylogenetically distant species, including A. eutrophus [X- nological assays revealed that cells of A. eutrophus, containing active MBH, produce the small and large subunits of the enzyme in two electrophoretically distinct conformations. It was suggested that the conversion of the two subunits into the catalytically active membrane-associated heterodimer is dependent on the function of accessory-gene products [12]. More recently, we described two classes of mutants with in-frame deletions in the eight MBH-linked accessory genes. Class-I mutants, affected in hoxM, hoxO and hoxQ, are totally devoid of MBH activity, whereas class-I1 mutants, harboring del...

We have recently established that the facultative phototrophic bacterium Rhodobacter capsulatus has two different pathways for reduction of the photooxidized reaction center during photosynthesis (F. E. Jenney and F. Daldal, EMBO J. 12:1283-1292, 1993; F. E. Jenney, R. C. Prince, and F. Daldal, Biochemistry 33:2496-2502, 1994). One pathway is via the well-characterized, water-soluble cytochrome c 2 (cyt c 2 ), and the other is via a novel membrane-associated c-type cytochrome named cyt c y . In this work, we probed the role of cyt c y in respiratory electron transport by isolating a set of R. capsulatus mutants lacking either cyt c 2 or cyt c y , in the presence or in the absence of a functional quinol oxidase-dependent alternate respiratory pathway. The growth and inhibitor sensitivity patterns of these mutants, their respiratory rates in the presence of specific inhibitors, and the oxidation-reduction kinetics of c-type cytochromes monitored under appropriate conditions demonstrated that cyt c y , like cyt c 2 , connects the bc 1 complex and the cyt c oxidase during respiratory electron transport. Whether cyt c 2 and cyt c y are the only electron carriers between these two energy-transducing membrane complexes of R. capsulatus is unknown.

The dorC gene of the dimethyl sulfoxide respiratory (dor) operon of Rhodobacter capsulatus encodes a pentaheme c-type cytochrome that is involved in electron transfer from ubiquinol to periplasmic dimethyl sulfoxide reductase. DorC was expressed as a C-terminal fusion to an 8-amino acid FLAG epitope and was purified from detergent-solubilized membranes by ion exchange chromatography and immunoaffinity chromatography. The DorC protein had a subunit M r ‫؍‬ 46,000, and pyridine hemochrome analysis indicated that it contained 5 mol heme c/mol DorC polypeptide, as predicted from the derived amino acid sequence of the dorC gene. The reduced form of DorC exhibited visible absorption maxima at 551.5 nm (␣-band), 522 nm (␤-band), and 419 nm (Soret band). Redox potentiometry of the heme centers of DorC identified five components (n ‫؍‬ 1) with midpoint potentials of ؊34, ؊128, ؊184, ؊185, and ؊276 mV. Despite the low redox potentials of the heme centers, DorC was reduced by duroquinol and was oxidized by dimethyl sulfoxide reductase.The ability to use Me 2 SO and trimethylamine-N-oxide (TMAO) 1 as electron acceptors is widespread among facultative aerobic bacteria and the organization of the Me 2 SO and TMAO respiratory chains is now well defined (1). In the Me 2 SO respiratory system of purple photosynthetic bacteria such as Rhodobacter capsulatus, electrons are transferred from primary dehydrogenases via the ubiquinone pool to a periplasmic Me 2 SO reductase (2). Recent sequence and mutational analysis of the Me 2 SO respiratory gene cluster from photosynthetic bacteria has identified a pentaheme c-type cytochrome (DorC) as the likely mediator of electron transfer from ubiquinol to Me 2 SO reductase (3).2 The TMAO respiratory (Tor) system of Escherichia coli (5) is very similar to the Dor system of R. capsulatus, and they differ from the Me 2 SO respiratory (Dms) system of E. coli. The Me 2 SO reductase (DmsABC) of E. coli can be purified as a menaquinol-oxidizing Me 2 SO reductase complex that lacks c-type cytochromes (6). Me 2 SO reductase from R. capsulatus has been purified as a monomeric protein containing a pterin molybdenum cofactor (Moco) as its only prosthetic group (7). This property of Me 2 SO reductase has many advantages for spectroscopic characterization of Moco, but a major deficiency has been the lack of a physiologically relevant electron donor. To overcome this problem we describe in this paper the purification and characterization of DorC.The derived amino acid sequence of Rhodobacter DorC indicated that it was related to members of the NirT class of tetraheme c-type cytochromes (3).2 However, DorC is predicted to be a pentaheme cytochrome with a fifth c-type heme binding motif in the C-terminal polypeptide, which is absent from the tetraheme members of the NirT class. Almost all of the members of the NirT class are involved in an anaerobic respiratory pathway, and their role appears to be to catalyze electron transfer from the Q-pool to a periplasmic terminal reductase (8). Although molecular geneti...

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Incubation of 3-d-old seedlings of Oryza sativa L. cv Arborio under anaerobic conditions, leads to a large increase in the titer of free putrescine while aerobic incubation causes a slight decrease. After 2 days, the putrescine level is about 2.5 times greater without oxygen than in air. The rice coleoptile also accumulates a large amount of bound putrescine and, to a lesser extent, spermidine and spermine (mainly as acid-soluble conjugates). Accumulation of conjugates in the roots is severely inhibited by the anaerobic treatment. Feeding experiments with labeled amino acids showed that anoxia stimulates the release of (14)CO(2) from tissues fed with [(14)C]arginine and that arginine is the precursor in putrescine biosynthesis. After 2 d of anoxia, the activity of arginine decarboxylase was 42% and 89% greater in coleoptile and root, respectively, than in the aerobic condition. The causes of the differences in polyamine metabolism in anoxic coleoptiles and roots are discussed.

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During 2020, the COVID-19 pandemic affected almost 10 8 individuals. Quite a number of vaccines against COVID-19 were therefore developed, and a few recently received authorization for emergency use. Overall, these vaccines target specific viral proteins by antibodies whose synthesis is directly elicited or indirectly triggered by nucleic acids coding for the desired targets. Among these targets, the receptor binding domain (RBD) of COVID-19 spike protein (SP) does frequently occur in the repertoire of candidate vaccines. However, the immunogenicity of RBD per se is limited by its low molecular mass, and by a structural rearrangement of full-length SP accompanied by the detachment of RBD. Here we show that the RBD of COVID-19 SP can be conveniently produced in Escherichia coli when fused to a fragment of CRM197, a variant of diphtheria toxin currently used for a number of conjugated vaccines. In particular, we show that the CRM197-RBD chimera solubilized from inclusion bodies can be refolded and purified to a state featuring the 5 native disulphide bonds of the parental proteins, the competence in binding angiotensin-converting enzyme 2, and a satisfactory stability at room temperature. Accordingly, our observations provide compulsory information for the development of a candidate vaccine directed against COVID-19.