Levers and Barriers to Vaccinate against COVID-19 in the Multicultural Context of French Guiana: A Qualitative Cross-Sectional Survey among Health Care Workers.

In French Guiana, a French overseas territory in South America facing a fourth wave of COVID-19, vaccination coverage is very low, both in the population and among health care workers (HCWs). Vaccine hesitancy concerned 35.7% of the latter in early 2021. The objective of this complementary study is to understand barriers and levers and to adapt messages to increase vaccination coverage among HCWs. We conducted a regional cross-sectional survey of HCWs with a questionnaire containing open-ended questions exploring factors associated with vaccine hesitancy and the needs to adapt the vaccination campaign in French Guiana. The discourses were analyzed using a qualitative approach based on grounded theory, with open coding of data by themes and construction of abstract categories. The analysis of the 357 responses collected from January to March 2021 reveals several trends. The ethical aspect of the HCWs' role emphasizes the importance of getting vaccinated themselves (to protect patients, to set an example...) and of vaccinating as many people as possible, including the most geographically or socially distant, such as undocumented migrants. However, some HCWs remain suspicious of the vaccine with concerns over the efficacy and side effects, of health institutions, and of the pharmaceutical industry. The role of fake news circulating on social networks has been widely discussed. Efforts to explain and convince HCWs must be continued in French Guiana using the identified levers to improve the acceptability of vaccination.

Attitudes towards the COVID-19 Vaccine and Willingness to Get Vaccinated among Healthcare Workers in French Guiana: The Influence of Geographical Origin.

Background: In the context of the global COVID-19 pandemic and the expansion of the more transmissible 20J/501Y.V3 (Gamma) variant of concern (VOC), mRNA vaccines have been made available in French Guiana, an overseas French territory in South America, from mid-January 2021. This study aimed to estimate the willingness to be vaccinated and the socio-demographic and motivational correlates among Health Care Workers (HCWs) in French Guiana. Methods: A cross-sectional survey was conducted from January 22 to March 26, 2021 among a sample of HCWs in French Guiana. They were asked about their willingness to get vaccinated against COVID-19 and vaccine hesitancy, vaccine uptake and vaccines attitudes. Factors associated with willingness to get vaccinated have been analyzed with ordinal logistic regression, using Stata software. Results: A total of 579 HCWs were interviewed, including 220 physicians and 200 nurses most often working in hospital (54%) or in the liberal sector (22%). Overall, 65.6% of respondents reported that they were willing or had already been vaccinated against COVID-19, while 24.3% of respondents reported that they did not want to get vaccinated against COVID-19 and 11.2% were unsure. HCWs were more willing to get vaccine if they were older, were worried about COVID-19 and were confident in the management of epidemic. Conversely, participants were less likely to have been vaccinated or willing to if they were nurses or of another non-medical profession, born in French Guiana, feared adverse effects, or if they did not trust pharmaceutical companies and management of the epidemic by authorities. Conclusion: Negative attitudes towards vaccines are a major public health concern among HCWs in French Guiana when considering the current active epidemic with Gamma VOC. General vaccine hesitancy and concerns about future side effects in particular represent important barriers. Low confidence in government and science are significant in COVID-19 vaccine refusal among non-medical staffs. Public health messaging with information on vaccine safety should be tailored to address these concerns. The specific challenges of HCWs from French Guiana must be taken into account.

An investigation of slow charge separation in a tyrosine M210 to tryptophan mutant of the Rhodobacter sphaeroides reaction center by femtosecond mid-infrared spectroscopy.

Energy and electron transfer in a tyrosine M210 to tryptophan (YM210W) mutant of the Rhodobacter sphaeroides reaction center (RC) were investigated through time-resolved visible pump/mid-infrared (mid-IR) probe spectroscopy at room temperature, with the aim to further characterize the primary charge separated states in the RC. This mutant is known to display slow and multi-exponential charge separation, and was used in earlier work to prove the existence of an alternative route for charge separation starting from the accessory bacteriochlorophyll in the active branch, B(L). The mutant RCs were excited at 860 nm (direct excitation of the primary donor (P) BChls (P(L)/P(M))), 600 nm (unselective excitation), 805 nm (direct excitation of both accessory bacteriochlorophyll cofactors B(L) and B(M)) and 795 nm (direct excitation of B(L)). Absorption changes associated with carbonyl (C=O) stretch vibrational modes of the cofactors and protein were recorded in the region between 1600 and 1775 cm(-1), and both a sequential analysis and simultaneous target analysis of the data were performed. The decay of P* in the YM210W mutant was multi-exponential with lifetimes of 29 and 63.5 ps. The decay of P(+)B(L)(-) state was approximately 10 times longer in the YM210W RC than in the R-26 RC (approximately 7 ps vs. approximately 0.7 ps), and in the mid-IR difference absorption spectrum of P(+)B(L)(-) the stretching frequency of the 9-keto C=O group of B(L) in the ground state was located around 1675-1680 cm(-1), consistent with the presence of a hydrogen bond donated by an adjacent water molecule. Excitation at 795 nm produced a small amount of B(L)*-driven charge separation, as assessed from the excitation wavelength dependence of the raw difference spectra recorded during the first few ps after excitation. This process led to the formation of P(+)B(L)(-). Only the relaxed form of the P(+)H(L)(-) radical pair was observed in the YM210W mutant, and the mid-IR difference absorption spectra of P(+)H(L)(-) and P(+)B(L)(-) showed a change in the relative amplitude of the P(L)(+) and P(M)(+) bands when compared to equivalent spectra for the R-26 RC. This indicates that the YM210W mutation causes an increased localization of the electron hole on the P(M) half of the dimer. The absorbance difference spectrum of P(+)H(L)(-) in the R-26 RC contains a feature attributable to a Stark shift of one or more amide C=O oscillators. This feature was shifted to lower frequency by approximately 5 cm(-1) in the YM210W RC, and consideration of the limited structural changes in this RC indicates that this feature arises from an amide C=O group in the immediate vicinity of the M210 residue, most probably that of the adjacent M209 amino acid.

Identification of the intermediate charge-separated state P+betaL- in a leucine M214 to histidine mutant of the Rhodobacter sphaeroides reaction center using femtosecond midinfrared spectroscopy.

Energy and electron transfer in a Leu M214 to His (LM214H) mutant of the Rhodobacter sphaeroides reaction center (RC) were investigated by applying time-resolved visible pump/midinfrared probe spectroscopy at room temperature. This mutant replacement of the Leu at position M214 resulted in the incorporation of a bacteriochlorophyll (BChl) in place of the native bacteriopheophytin in the L-branch of cofactors (denoted betaL). Purified LM214H RCs were excited at 600 nm (unselective excitation), at 800 nm (direct excitation of the monomeric BChl cofactors B(L) and B(M)), and at 860 nm (direct excitation of the primary donor (P) BChl pair (P(L)/P(M))). Absorption changes associated with carbonyl (C=O) stretch vibrational modes (9-keto, 10a-ester, and 2a-acetyl) of the cofactors and of the protein were recorded in the region between 1600 cm(-1) and 1770 cm(-1), and the data were subjected to both a sequential analysis and a simultaneous target analysis. After photoexcitation of the LM214H RC, P* decayed on a timescale of approximately 6.3 ps to P+BL-. The decay of P+BL- occurred with a lifetime of approximately 2 ps, approximately 3 times slower than that observed in wild-type and R-26 RCs (approximately 0.7 ps). Further electron transfer to the betaL BChl resulted in formation of the P+betaL- state, and its infrared absorbance difference spectrum is reported for the first time, to our knowledge. The fs midinfrared spectra of P+BL- and P+betaL- showed clear differences related to the different environments of the two BChls in the mutant RC.

Coupling of electron transfer to proton uptake at the Q(B) site of the bacterial reaction center: a perspective from FTIR difference spectroscopy.

FTIR difference spectroscopy provides a unique approach to study directly protonation/deprotonation events of carboxylic acids involved in the photochemical cycle of membrane proteins, such as the bacterial photosynthetic reaction center (RC). In this work, we review the data obtained by light-induced FTIR difference spectroscopy on the first electron transfer to the secondary quinone Q(B) in native RCs and a series of mutant RCs. We first examine the approach of isotope-edited FTIR spectroscopy to investigate the binding site of Q(B). This method provides highly specific IR vibrational fingerprints of the bonding interactions of the carbonyls of Q(B) and Q(B)(-) with the protein. The same isotope-edited IR fingerprints for the carbonyls of neutral Q(B) have been observed for native Rhodobacter sphaeroides RCs and several mutant RCs at the Pro-L209, Ala-M260, or Glu-L212/Asp-L213 sites, for which X-ray crystallography has found the quinone in the proximal position. It is concluded that at room temperature Q(B) occupies a single binding site that fits well the description of the proximal site derived from X-ray crystallography and that the conformational gate limiting the rate of the first electron transfer from Q(A)(-)Q(B) to Q(A)Q(B)(-) cannot be the movement of Q(B) from its distal to proximal site. Possible alternative gating mechanisms are discussed. In a second part, we review the contribution of the various experimental measurements, theoretical calculations, and molecular dynamics simulations which have been actively conducted to propose which amino acid side chains near Q(B) could be proton donors/acceptors. Further, we show how FTIR spectroscopy of mutant RCs has directly allowed several carboxylic acids involved in proton uptake upon first electron transfer to Q(B) to be identified. Owing to the importance of a number of residues for high efficiency of coupled electron transfer reactions, the photoreduction of Q(B) was studied in a series of single mutant RCs at Asp-L213, Asp-L210, Asp-M17, Glu-L212, Glu-H173, as well as combinations of these mutations in double and triple mutant RCs. The same protonation pattern was observed in the 1760-1700 cm(-1) region of the Q(B)(-)/Q(B) spectra of native and several mutant (DN-L213, DN-L210, DN-M17, EQ-H173) RCs. However, it was drastically modified in spectra of mutants lacking Glu at L212. The main conclusion of this work is that in native RCs from Rb. sphaeroides, Glu-L212 is the only carboxylic acid residue that contributes to proton uptake at all pH values (from pH 4 to pH 11) in response to the formation of Q(B)(-). Another important result is that the residues Asp-L213, Asp-L210, Asp-M17, and Glu-H173 are mostly ionized in the Q(B) state at neutral pH and do not significantly change their protonation state upon Q(B)(-) formation. In contrast, interchanging Asp and Glu at L212 and L213 (i.e., in the so-called swap mutant) led to the identification of a novel protonation pattern of carboxylic acids: at least four individual carboxylic acids were affected by Q(B) reduction. The pH dependence of IR carboxylic signals in the swap mutant demonstrates that protonation of Glu-L213 occurred at pH >5 whereas that of Asp-L212 occurred over the entire pH range from 8 to 4. In native RCs from Rhodobacter sphaeroides, a broad positive IR continuum around 2600 cm(-1) in the Q(B)(-)/Q(B) steady-state FTIR spectrum in (1)H(2)O was assigned to delocalized proton(s) in a highly polarizable hydrogen-bonded network. The possible relation of the IR continuum band to the carboxylic acid residues and to bound water molecules involved in the proton transfer pathway was investigated by testing the robustness of this band to different mutations of acids. The presence of the band is not correlated with the localization of the proton on Glu-L212. The largest changes of the IR continuum were observed in single and double mutant RCs where Asp-L213 is not present. It is proposed that the changes observed in the mutant RCs with respect to native RCs reflect the specific role of bound protonated water molecule(s) located in the vicinity of Asp-L213 and undergoing hydrogen-bond changes in the network.

Primary charge separation in the photosystem II core from Synechocystis: a comparison of femtosecond visible/midinfrared pump-probe spectra of wild-type and two P680 mutants.

It is now quite well accepted that charge separation in PS2 reaction centers starts predominantly from the accessory chlorophyll B(A) and not from the special pair P(680). To identify spectral signatures of B(A,) and to further clarify the process of primary charge separation, we compared the femtosecond-infrared pump-probe spectra of the wild-type (WT) PS2 core complex from the cyanobacterium Synechocystis sp. PCC 6803 with those of two mutants in which the histidine residue axially coordinated to P(B) (D2-His(197)) has been changed to Ala or Gln. By analogy with the structure of purple bacterial reaction centers, the mutated histidine is proposed to be indirectly H-bonded to the C(9)=O carbonyl of the putative primary donor B(A) through a water molecule. The constructed mutations are thus expected to perturb the vibrational properties of B(A) by modifying the hydrogen bond strength, possibly by displacing the H-bonded water molecule, and to modify the electronic properties and the charge localization of the oxidized donor P(680)(+). Analysis of steady-state light-induced Fourier transform infrared difference spectra of the WT and the D2-His(197)Ala mutant indeed shows that a modification of the axially coordinating ligand to P(B) induces a charge redistribution of P(680)(+). In addition, a comparison of the time-resolved visible/midinfrared spectra of the WT and mutants has allowed us to investigate the changes in the kinetics of primary charge separation induced by the mutations and to propose a band assignment identifying the characteristic vibrations of B(A).

Identification of the first steps in charge separation in bacterial photosynthetic reaction centers of Rhodobacter sphaeroides by ultrafast mid-infrared spectroscopy: electron transfer and protein dynamics.

Time-resolved visible pump/mid-infrared (mid-IR) probe spectroscopy in the region between 1600 and 1800 cm(-1) was used to investigate electron transfer, radical pair relaxation, and protein relaxation at room temperature in the Rhodobacter sphaeroides reaction center (RC). Wild-type RCs both with and without the quinone electron acceptor Q(A), were excited at 600 nm (nonselective excitation), 800 nm (direct excitation of the monomeric bacteriochlorophyll (BChl) cofactors), and 860 nm (direct excitation of the dimer of primary donor (P) BChls (P(L)/P(M))). The region between 1600 and 1800 cm(-1) encompasses absorption changes associated with carbonyl (C=O) stretch vibrational modes of the cofactors and protein. After photoexcitation of the RC the primary electron donor P excited singlet state (P*) decayed on a timescale of 3.7 ps to the state P(+)B(L)(-) (where B(L) is the accessory BChl electron acceptor). This is the first report of the mid-IR absorption spectrum of P(+)B(L)(-); the difference spectrum indicates that the 9-keto C=O stretch of B(L) is located around 1670-1680 cm(-1). After subsequent electron transfer to the bacteriopheophytin H(L) in approximately 1 ps, the state P(+)H(L)(-) was formed. A sequential analysis and simultaneous target analysis of the data showed a relaxation of the P(+)H(L)(-) radical pair on the approximately 20 ps timescale, accompanied by a change in the relative ratio of the P(L)(+) and P(M)(+) bands and by a minor change in the band amplitude at 1640 cm(-1) that may be tentatively ascribed to the response of an amide C=O to the radical pair formation. We conclude that the drop in free energy associated with the relaxation of P(+)H(L)(-) is due to an increased localization of the electron hole on the P(L) half of the dimer and a further consequence is a reduction in the electrical field causing the Stark shift of one or more amide C=O oscillators.

The interplay between nitric oxide and peroxiredoxins.

Peroxiredoxins participate in the antioxidant response by reducing H(2)O(2), organic peroxides and peroxynitrite. Peroxiredoxins have a conserved NH(2)-terminal cysteine residue that is oxidized to sulfenic acid during catalysis of peroxide reduction. In eukaryotes, the sulfenic acid can be further oxidized to a sulfinic acid. Resulting inactivation of peroxiredoxins favors H(2)O(2) signaling but may eventually result in oxidative stress. Interestingly, it has recently been shown that overoxidized peroxiredoxins progressively recover activity owing to sulfiredoxin, an enzyme recently characterized in yeast and mammals. This reversible peroxide-sensitive switch represents a new type of regulation that controls reactive oxygen species-mediated cytoxicity and signaling. This report presents a brief overview of the regulation by peroxiredoxins of the messenger function of H(2)O(2) and comments on the results of recent studies that addressed the consequence of nitric oxide production on both expression and redox state of peroxiredoxins in various physiopathological processes including macrophage immunostimulation, the response of dopaminergic neurons to N-methyl-d-aspartate-stimulation and the plant hypersensitive response.

Replacement or exclusion of the B-branch bacteriopheophytin in the purple bacterial reaction centre: the H(B) cofactor is not required for assembly or core function of the Rhodobacter sphaeroides complex.

All of the membrane-embedded cofactors of the purple bacterial reaction centre have well-defined functional or structural roles, with the exception of the bacteriopheophytin (H(B)) located approximately half-way across the membrane on the so-called inactive- or B-branch of cofactors. Sequence alignments indicate that this bacteriochlorin cofactor is a conserved feature of purple bacterial reaction centres, and a pheophytin is also found at this position in the Photosystem-II reaction centre. Possible structural or functional consequences of replacing the H(B) bacteriopheophytin by bacteriochlorophyll were investigated in the Rhodobacter sphaeroides reaction centre through mutagenesis of residue Leu L185 to His (LL185H). Results from absorbance spectroscopy indicated that the LL185H mutant assembled with a bacteriochlorophyll at the H(B) position, but this did not affect the capacity of the reaction centre to support photosynthetic growth, or change the kinetics of charge separation along the A-branch of cofactors. It was also found that mutation of residue Ala M149 to Trp (AM149W) caused the reaction centre to assemble without an H(B) bacteriochlorin, demonstrating that this cofactor is not required for correct assembly of the reaction centre. The absence of a cofactor at this position did not affect the capacity of the reaction centre to support photosynthetic growth, or the kinetics of A-branch electron transfer. A combination of X-ray crystallography and FTIR difference spectroscopy confirmed that the H(B) cofactor was absent in the AM149W mutant, and that this had not produced any significant disturbance of the adjacent ubiquinol reductase (Q(B)) site. The data are discussed with respect to possible functional roles of the H(B) bacteriopheophytin, and we conclude that the reason(s) for conservation of a bacteriopheophytin cofactor at this position in purple bacterial reaction centres are likely to be different from those underlying conservation of a pheophytin at the analogous position in Photosystem-II.

ATR-FTIR difference spectroscopy of the P(M) intermediate of bovine cytochrome c oxidase.

Perfusion-induced attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to investigate changes induced in protein and cofactors of bovine cytochrome c oxidase when it was converted from the oxidised state to the catalytic P(M) intermediate. The transition was induced in a film of detergent-depleted 'fast' oxidase with a buffer containing CO and O(2). The extent of formation of the P(M) state was quantitated simultaneously by monitoring formation of its characteristic 607-nm band with a scanned visible beam reflected off the top surface of the prism. The P(M) minus O FTIR difference spectrum is distinctly different from the redox spectra reported to date and includes features that can be assigned to changes of haem a(3) and surrounding protein. Tentative assignments are made based on vibrational data of related proteins and model compounds.

The Lhca antenna complexes of higher plants photosystem I.

The Lhca antenna complexes of photosystem I (PSI) have been characterized by comparison of native and recombinant preparations. Eight Lhca polypeptides have been found to be all organized as dimers in the PSI-LHCI complex. The red emission fluorescence is associated not only with Lhca1-4 heterodimer, but also with dimers containing Lhca2 and/or Lhca3 complexes. Reconstitution of Lhca1 and Lhca4 monomers as well as of the Lhca1-4 dimer in vitro was obtained. The biochemical and spectroscopic features of these three complexes are reported. The monomers Lhca1 and Lhca4 bind 10 Chls each, while the Chl a/b ratio is lower in Lhca4 as compared to Lhca1. Three carotenoid binding sites have been found in Lhca1, while only two are present in Lhca4. Both complexes contain lutein and violaxanthin while beta-carotene is selectively bound to the Lhca1-4 dimer in substoichiometric amounts upon dimerization. Spectral analysis revealed the presence of low energy absorption forms in Lhca1 previously thought to be exclusively associated with Lhca4. It is shown that the process of dimerization changes the spectroscopic properties of some chromophores and increases the amplitude of the red absorption tail of the complexes. The origin of these spectroscopic features is discussed.

Rapid-scan Fourier transform infrared spectroscopy shows coupling of GLu-L212 protonation and electron transfer to Q(B) in Rhodobacter sphaeroides reaction centers.

Rapid-scan Fourier transform infrared (FTIR) difference spectroscopy was used to investigate the electron transfer reaction Q(A-)Q(B)-->Q(A)Q(B-) (k(AB)(1)) in mutant reaction centers of Rhodobacter sphaeroides, where Asp-L210 and/or Asp-M17 have been replaced with Asn. Mutation of both residues decreases drastically k(AB)(1)), attributed to slow proton transfer to Glu-L212, which becomes rate limiting for electron transfer to Q(B) [M.L. Paddock et al., Biochemistry 40 (2001) 6893]. In the double mutant, the FTIR difference spectrum recorded during the time window 4-29 ms following a flash showed peaks at 1670 (-), 1601 (-) and 1467 (+) cm(-1), characteristic of Q(A) reduction. The time evolution of the spectra shows reoxidation of Q(A-) and concomitant reduction of Q(B) with a kinetics of about 40 ms. In native reaction centers and in both single mutants, formation of Q(B-) occurs much faster than in the double mutant. Within the time resolution of the technique, protonation of Glu-L212, as characterized by an absorption increase at 1728 cm(-1) [E. Nabedryk et al., Biochemistry 34 (1995) 14722], was found to proceed with the same kinetics as reduction of Q(B) in all samples. These rapid-scan FTIR results support the model of proton uptake being rate limiting for the first electron transfer from Q(A-) to Q(B) and the identification of Glu-L212 as the main proton acceptor in the state Q(A)Q(B-).

Characterization of the bonding interactions of Q(B) upon photoreduction via A-branch or B-branch electron transfer in mutant reaction centers from Rhodobacter sphaeroides.

In Rhodobacter sphaeroides reaction centers (RCs) containing the mutation Ala M260 to Trp (AM260W), transmembrane electron transfer along the full-length of the A-branch of cofactors is prevented by the loss of the Q(A) ubiquinone, but it is possible to generate the radical pair P(+)H(A)(-) by A-branch electron transfer or the radical pair P(+)Q(B)(-) by B-branch electron transfer. In the present study, FTIR spectroscopy was used to provide direct evidence for the complete absence of the Q(A) ubiquinone in mutant RCs with the AM260W mutation. Light-induced FTIR difference spectroscopy of isolated RCs was also used to probe the neutral Q(B) and the semiquinone Q(B)(-) states in two B-branch active mutants, a double AM260W-LM214H mutant, denoted WH, and a quadruple mutant, denoted WAAH, in which the AM260W, LM214H, and EL212A-DL213A mutations were combined. The data were compared to those obtained with wild-type (Wt) RCs and the double EL212A-DL213A (denoted AA) mutant which exhibit the usual A-branch electron transfer to Q(B). The Q(B)(-)/Q(B) spectrum of the WH mutant is very close to that of Wt RCs indicating similar bonding interactions of Q(B) and Q(B)(-) with the protein in both RCs. The Q(B)(-)/Q(B) spectra of the AA and WAAH mutants are also closely related to one another, but are very different to that of the Wt complex. Isotope-edited IR fingerprint spectra were obtained for the AA and WAAH mutants reconstituted with site-specific (13)C-labeled ubiquinone. Whilst perturbations of the interactions of the semiquinone Q(B)(-) with the protein are observed in the AA and WAAH mutants, the FTIR data show that the bonding interaction of neutral Q(B) in these two mutants are essentially the same as those for Wt RCs. Therefore, it is concluded that Q(B) occupies the same binding position proximal to the non-heme iron prior to reduction by either A-branch or B-branch electron transfer.

Tuning of the optical and electrochemical properties of the primary donor bacteriochlorophylls in the reaction centre from Rhodobacter sphaeroides: spectroscopy and structure.

A series of mutations have been introduced at residue 168 of the L-subunit of the reaction centre from Rhodobacter sphaeroides. In the wild-type reaction centre, residue His L168 donates a strong hydrogen bond to the acetyl carbonyl group of one of the pair of bacteriochlorophylls (BChl) that constitutes the primary donor of electrons. Mutation of His L168 to Phe or Leu causes a large decrease in the mid-point redox potential of the primary electron donor, consistent with removal of this strong hydrogen bond. Mutations to Lys, Asp and Arg cause smaller decreases in redox potential, indicative of the presence of weak hydrogen bond and/or an electrostatic effect of the polar residue. A spectroscopic analysis of the mutant complexes suggests that replacement of the wild-type His residue causes a decrease in the strength of the coupling between the two primary donor bacteriochlorophylls. The X-ray crystal structure of the mutant in which His L168 has been replaced by Phe (HL168F) was determined to a resolution of 2.5 A, and the structural model of the HL168F mutant was compared with that of the wild-type complex. The mutation causes a shift in the position of the primary donor bacteriochlorophyll that is adjacent to residue L168, and also affects the conformation of the acetyl carbonyl group of this bacteriochlorophyll. This conformational change constitutes an approximately 27 degrees through-plane rotation, rather than the large into-plane rotation that has been widely discussed in the context of the HL168F mutation. The possible structural basis of the altered spectroscopic properties of the HL168F mutant reaction centre is discussed, as is the relevance of the X-ray crystal structure of the HL168F mutant to the possible structures of the remaining mutant complexes.

Photoreduction of the quinone pool in the bacterial photosynthetic membrane: identification of infrared marker bands for quinol formation.

The photoreduction of the quinone (Q) pool in the photosynthetic membrane of the purple bacterium Rhodobacter sphaeroides was investigated by steady-state and time-resolved Fourier transform infrared difference spectroscopy. The results are consistent with the existence of a homogeneous Q pool inside the chromatophore membrane, with a size of around 20 Q molecules per reaction center. IR marker bands for the quinone/quinol (Q/QH(2)) redox couple were recognized. QH(2) bands are identified at 1491, 1470, 1433 and 1388-1375 cm(-1). The 1491 cm(-1) band, which is sensitive to (1)H/(2)H exchange, is assigned to a C-C ring mode coupled to a C-OH mode. A feature at approximately 1743/1720 cm(-1) is tentatively related to a perturbation of the carbonyl modes of phospholipid head groups induced by QH(2) formation. Complex conformational changes of the protein in the amide I and II spectral ranges are also apparent during reduction and reoxidation of the Q pool.

Time-resolved step-scan FTIR investigation on the primary donor of the reaction center from the green sulfur bacterium Chlorobium tepidum.

The vibrational properties of the primary donor P(840) in the reaction center (RC) of the green sulfur bacterium Chlorobium tepidum and its interactions with the surrounding protein environment have been investigated by Fourier transform infrared (FTIR) difference spectroscopy at cryogenic temperatures. By using the step-scan technique with a time resolution of 5 mus on RCs that had been depleted of the iron-sulfur electron acceptors, the formation and decay of the triplet state (3)P(840) have been followed in infrared for the first time. The (3)P(840)/P(840) FTIR difference spectrum is compared to the P(840) (+)/P(840) FTIR difference spectrum measured under identical conditions on untreated RCs and recorded with the same step-scan set-up. The latter P(840) (+)/P(840) difference spectrum is essentially the same as those measured under steady-state conditions using the more conventional continuous illumination method. Comparison of the (3)P(840)/P(840) and P(840) (+)/P(840) spectra provides unambiguous assignment of the vibration of the 9-keto C=O group(s) of P(840) at 1684 cm(-1) as the only common negative band in the two spectra. This frequency corresponds to carbonyl group(s) free from hydrogen bonding interactions. The obtained results are discussed in the framework of the structure and photochemistry of the primary donor P(840).

Attenuated total reflection Fourier transform infrared spectroscopy of redox transitions in photosynthetic reaction centers: comparison of perfusion- and light-induced difference spectra.

Chemically induced Fourier transform infrared difference spectra associated with redox transitions of several primary electron donors and acceptors in photosynthetic reaction centers (RCs) have been compared with the light-induced FTIR difference spectra involving the same cofactors. The RCs are deposited on an attenuated total reflection (ATR) prism and form a film that is enclosed in a flow cell. Redox transitions in the film of RCs can be repetitively induced either by perfusion of buffers poised at different redox potentials or by illumination. The perfusion-induced ATR-FTIR difference spectra for the oxidation of the primary electron donor P in the RCs of the purple bacteria Rb. sphaeroides and Rp. viridis and P700 in the photosystem 1 of Synechocystis 6803, as well as the Q(A)/Q(A) transition of the quinone acceptor (Q(A)) in Rb. sphaeroides RCs are reported for the first time. They are compared with the light-induced ATR-FTIR difference spectra P+Q(A)/PQ(A) for the RCs of Rb. sphaeroides and P700+/P700 for photosystem 1. It is shown that the perfusion-induced and light-induced ATR-FTIR difference spectra recorded on the same RC film display identical signal to noise ratios when they are measured under comparable conditions. The ATR-FTIR difference spectra are very similar to the equivalent FTIR difference spectra previously recorded upon photochemical or electrochemical excitation of these RCs in the more conventional transmission mode. The ATR-FTIR technique requires a smaller amount of sample compared with transmission FTIR and allows precise control of the aqueous environment of the RC films.

Peroxiredoxin post-translational modifications by redox messengers.

Peroxiredoxins (Prxs) are a family of thiol peroxidases that participate in hydroperoxide detoxification and regulates H2O2 signaling. In mammals, the four typical 2-Cys Prxs (Prxs 1, 2, 3 and 4) are known to regulate H2O2-mediated intracellular signaling. The 2 catalytic cysteines of 2-Cys Prxs, the so-called peroxidatic and resolving cysteines, are regulatory switches that are prone to react with redox signaling molecules. We investigated the respective modifications induced by H2O2, NO and H2S in the murine macrophage cell line RAW264.7 by mass spectrometry and immunoblotting after separating 2-Cys Prxs by one-dimensional or two-dimensional PAGE. We found that H2S, unlike NO, does not prevent H2O2-mediated sulfinylation of 2-Cys Prxs and that Prx2 is more sensitive to NO-mediated protection against sulfinylation by peroxides. We also observed that cells exposed to exogenous NO, released by Cys-SNO or DETA-NO, or producing NO upon stimulation by IFN-γ and LPS, present an acidic form of Prx1 whose modification is consistent with S-homocysteinylation of its peroxidatic cysteine.

Nitric oxide activates an Nrf2/sulfiredoxin antioxidant pathway in macrophages.

Peroxiredoxins (Prx's) are a family of peroxidases that maintain thiol homeostasis by catalyzing the reduction of organic hydroperoxides, H2O2, and peroxynitrite. Under conditions of oxidative stress, eukaryotic Prx's can be inactivated by the substrate-dependent oxidation of the catalytic cysteine to sulfinic acid, which may regulate the intracellular messenger function of H2O2. A small redox protein, sulfiredoxin (Srx), conserved only in eukaryotes, has been shown to reduce sulfinylated 2-Cys Prx's, adding to the complexity of the H2O2 signaling network. In this study, we addressed the regulation of Srx expression in immunostimulated primary macrophages that produce both reactive oxygen species (ROS) and nitric oxide (NO(•)). We present genetic evidence that NO-mediated Srx up-regulation is mediated by the transcription factor nuclear factor erythroid 2-related factor (Nrf2). We also show that the NO(•)/Srx pathway inhibits generation of ROS. These results reveal a link between innate immunity and H2O2 signaling. We propose that an NO(•)/Nrf2/Srx pathway participates in the maintenance of redox homeostasis in cytokine-activated macrophages and other inflammatory settings.

Signaling events leading to peroxiredoxin 5 up-regulation in immunostimulated macrophages.

Peroxiredoxins (PRXs) are thiol peroxidases associated with many cellular functions including proliferation, cell cycle, apoptosis, and differentiation. There is also increasing evidence that these ubiquitous antioxidant enzymes control H(2)O(2) signaling in eukaryotes. Here, we provide evidence that the LPS/TLR4 and the Th1 cytokine IFN-gamma pathways induce expression of PRX5, a potent peroxide and peroxynitrite reductase, in primary macrophages. Furthermore, deletion of TRIF, MyD88, or type I IFN receptor revealed that the LPS/TLR4-dependent increase in PRX5 expression is mediated by a TRIF-dependent/IFN-beta-independent pathway. IFN-gamma-dependent induction of the PRX5 gene was markedly reduced in MyD88(-/-) and TNF(-/-) macrophages. Moreover, addition of exogenous TNF allowed the recovery of full PRX5 expression in both MyD88(-/-) and TNF(-/-) cells stimulated with IFN-gamma, suggesting that basal TNF produced in an MyD88-dependent manner contributes to PRX5 induction. Downstream of the TLR pathways, we have explored the role of MAPK activation and found that p38 and JNK mainly contribute to PRX5 up-regulation in immunostimulated macrophages. Expression of PRX5 is thus responsive to innate immunity signals, and we propose that PRX5 is an additional host defense weapon of activated macrophages.