[Prevalence of allergic rhinitis and associated factors in Mexican students. A multicenter study]. / Prevalencia de rinitis alérgica y factores asociados en estudiantes mexicanos. Un estudio multicéntrico.

OBJECTIVE: To determine the prevalence of allergic rhinitis and its associated factors in students from several Mexican states. METHODS: A cross-sectional study was conducted in order to identify the factors that are associated with the prevalence of allergic rhinitis. The "Allergic Rhinitis Diagnostic Questionnaire for Epidemiological Studies" was used, together with another questionnaire, to identify risk factors in allergic rhinitis. RESULTS: 11381 students of 12-24 years of age were included; the prevalence of allergic rhinitis was of 18 % (CI 95 % = 11.4-24.6), with predominance in females (60%). The factors associated with allergic rhinitis were: heredity, (OR = 2-4, p < 0.0001), respiratory infections, (OR = 2-4.6, p < 0.0001); areas with humidity at home (OR= 1.5 - 1.9, p < 0.0001), and being female (OR = 1.7 - 2.4, p < 0.002). The use of tobacco, the educational level and vehicular traffic, as well as carpeting and the use of scented disinfectants, showed no association. CONCLUSIONS: The factors associated with allergic rhinitis in students were; heredity, respiratory infections, humidity in house walls, and being female.

Objetivo: Determinar la prevalencia de rinitis alérgica y sus factores asociados en estudiantes de varios estados de la República Mexicana. Métodos: Se realizó un estudio transversal para identificar los factores asociados con la prevalencia de rinitis alérgica. Se utilizó el Cuestionario Diagnóstico de Rinitis Alérgica para Estudios Epidemiológicos y un cuestionario para identificar los factores de riesgo en rinitis alérgica. Resultados: Se incluyeron 11381 estudiantes de 12 a 24 años; la prevalencia de rinitis alérgica fue 18 % (IC 95 % = 11.4-24.6), con un predominio del sexo femenino (60 %). Los factores asociados con rinitis alérgica fueron herencia (RM = 2-4, p < 0.0001), infecciones respiratorias (RM = 2-4.6, p < 0.0001), zonas de humedad en casa (RM = 1.5-1.9, p < 0.0001), ser mujer (RM = 1.7-2.4, p < 0.002). El tabaquismo, el nivel educativo, el tráfico vehicular, la alfombra y el uso de desinfectantes aromatizados no mostraron asociación. Conclusiones: Los factores asociados con rinitis alérgica en estudiantes fueron la herencia, las infecciones respiratorias, la humedad en paredes de la casa y el sexo femenino.

The characterization of asthma with blood eosinophilia in adults in Latin America.

Objective: To identify and characterize asthma with blood eosinophilia in adults. Methods: This cross-sectional study consisted of 164 asthma patients, aged 18 years or older. Multivariate analyses by logistic regression were performed to identify clinical characteristics and biomarkers associated with asthma with blood eosinophilia (defined as asthma and a peripheral blood eosinophil count ≥400 cells/mm3). To evaluate the diagnostic accuracy of these biomarkers, the sensitivity, specificity and predictive values were calculated. Additionally, the area under the receiver operating characteristic (ROC) curve (AUC) was estimated for each biomarker. Results: Overall, 37.8% (95%CI: 30.7-45.4%) of asthma patients had blood eosinophilia. The following factors were associated with this characteristic: patient age <50 years (OR 3.25; 95% CI: 1.33-7.94), a serum level of IgE ≥300 UI/mL (OR 2.32; 95%CI: 1.14-4.75), and an Asthma Control Test (ACT) score <20 points (OR 3.10; 95%CI: 1.35-4.75); asthma with blood eosinophilia was also associated with a baseline FEV1/FVC <70% (OR 2.68; 95%CI: 1.28-5.59). On the other hand, age <50 years and ACT score <20 showed the highest sensitivity (above 80% each). Serum IgE level ≥300 UI/mL had the highest specificity (almost 68%). Finally, those with an ACT score <20 had the highest AUC (68%). Conclusions: In our study population, one-third of asthmatic adults had asthma with blood eosinophilia. Furthermore, the prevalence was greater in those ≤50 years of age; these patients experienced more severe, more poorly controlled asthma and had higher total serum IgE levels.

Is suicidal ideation associated with allergic asthma and allergic rhinitis?

OBJECTIVE: To investigate whether there is an association between suicidal ideation (SI) and allergic diseases in adults. METHODS: This was a comparative cross-sectional study involving individuals ranging from 20 to 50 years of age recruited from a university hospital in the city of Guadalajara, Mexico. We included patients with a confirmed diagnosis of allergic asthma, those with a confirmed diagnosis of allergic rhinitis, and healthy controls. All subjects completed the Beck Depression Inventory-II (BDI-II), which includes an item that evaluates the presence of suicidal thoughts or desires within the last two weeks, in order to identify SI. RESULTS: The sample comprised 115 patients with allergic asthma, 111 patients with allergic rhinitis, and 96 healthy controls. The number of individuals identified with SI in the three groups were, respectively, 17 (14.8%), 13 (11.7%), and 8 (8.3%). Regarding the presence of SI, no statistically significant association was found in the allergic asthma group (OR = 1.98; 95% CI: 0.78-4.64; p = 0.154) or in the allergic rhinitis group (OR = 1.46; 95% CI: 0.58-3.68; p = 0.424) when they were compared with the control group. However, the presence of depression was associated with SI in the three groups: allergic asthma (OR = 12.36; 95% CI: 2.67-57.15; p = 0.001); allergic rhinitis (OR = 6.20; 95% CI: 1.66-23.14; p = 0.006); and control (OR = 21.0; 95% CI: 3.75-117.36; p < 0,001). CONCLUSIONS: In comparison with the control group, no association was found between SI and the groups with allergic diseases. In contrast, there was association between SI and depression in the three groups.

Is suicidal ideation associated with allergic asthma and allergic rhinitis? / Ideação suicida apresenta associação com asma e rinite alérgicas?

ABSTRACT Objective: To investigate whether there is an association between suicidal ideation (SI) and allergic diseases in adults. Methods: This was a comparative cross-sectional study involving individuals ranging from 20 to 50 years of age recruited from a university hospital in the city of Guadalajara, Mexico. We included patients with a confirmed diagnosis of allergic asthma, those with a confirmed diagnosis of allergic rhinitis, and healthy controls. All subjects completed the Beck Depression Inventory-II (BDI-II), which includes an item that evaluates the presence of suicidal thoughts or desires within the last two weeks, in order to identify SI. Results: The sample comprised 115 patients with allergic asthma, 111 patients with allergic rhinitis, and 96 healthy controls. The number of individuals identified with SI in the three groups were, respectively, 17 (14.8%), 13 (11.7%), and 8 (8.3%). Regarding the presence of SI, no statistically significant association was found in the allergic asthma group (OR = 1.98; 95% CI: 0.78-4.64; p = 0.154) or in the allergic rhinitis group (OR = 1.46; 95% CI: 0.58-3.68; p = 0.424) when they were compared with the control group. However, the presence of depression was associated with SI in the three groups: allergic asthma (OR = 12.36; 95% CI: 2.67-57.15; p = 0.001); allergic rhinitis (OR = 6.20; 95% CI: 1.66-23.14; p = 0.006); and control (OR = 21.0; 95% CI: 3.75-117.36; p < 0,001). Conclusions: In comparison with the control group, no association was found between SI and the groups with allergic diseases. In contrast, there was association between SI and depression in the three groups.

RESUMO Objetivo: Investigar se existe associação entre ideação suicida (IS) e doenças alérgicas em adultos. Métodos: Estudo transversal comparativo envolvendo indivíduos com idade entre 20 e 50 anos recrutados em um hospital universitário da cidade de Guadalajara, México. Foram incluídos pacientes com diagnóstico confirmado de asma alérgica, aqueles com diagnóstico confirmado de rinite alérgica e controles saudáveis. Todos os sujeitos preencheram o Beck Depression Inventory-II (BDI-II), que inclui um item que avalia a presença de pensamentos ou desejos suicidas nas últimas duas semanas, a fim de identificar IS. Resultados: A amostra foi composta por 115 pacientes com asma alérgica, 111 pacientes com rinite alérgica e 96 controles saudáveis. O número de indivíduos identificados com IS nos três grupos foi de 17 (14,8%), 13 (11,7%) e 8 (8,3%), respectivamente. Quanto à presença de IS, não foi encontrada associação estatisticamente significativa no grupo asma alérgica (OR = 1,98; IC95%: 0,78-4,64; p = 0,154) ou no grupo rinite alérgica (OR = 1,46; IC95%: 0,58-3,68; p = 0,424) quando os mesmos foram comparados com o grupo controle. No entanto, a presença de depressão apresentou associação com IS nos três grupos: asma alérgica (OR = 12,36; IC95%: 2,67-57,15; p = 0,001); rinite alérgica (OR = 6,20; IC95%: 1,66-23,14; p = 0,006); e controle (OR = 21,0; IC95%: 3,75-117,36; p < 0,001). Conclusões: Em comparação com o grupo controle, não foi encontrada associação entre IS e os grupos com doenças alérgicas. Por outro lado, houve associação entre IS e depressão nos três grupos.

[Prevalence of vitamin D insufficiency and deficiency in Mexican adults with allergic asthma]. / Prevalencia de insuficiencia y deficiencia de vitamina D en adultos mexicanos con asma alérgica.

BACKGROUND: Hypovitaminosis D has been associated with various chronic diseases such as infections, autoimmune diseases, chronic obstructive pulmonary disease, cancer and asthma Objective: The objective at hand is to determine the prevalence of vitamin D (VD) insufficiency and deficiency in adults with allergic asthma. OBJECTIVE: Objective: The objective at hand is to determine the prevalence of vitamin D (VD) insufficiency and deficiency in adults with allergic asthma. METHODS: Through a cross-sectional study, we analyzed corresponding data amongst 135 patients. VD concentration was categorized as sufficient (≥ 30 ng/mL), insufficient (21-29 ng/mL), and deficient (≤ 20 ng/mL). The level of VD deficiency was measured through chemo-luminescence. We estimated the prevalence of VD alterations and their respective confidence intervals at 95 % (CI 95 %). RESULTS: Within the analyzed population, there were 99/135 women (73.3 %); the mean age was 34.5 ± 10.3 years. The mean concentration of VD was 17.9 ± 6.9 ng/mL and the median was 17 ng/mL. The prevalence of VD insufficiency and deficiency was 25.2 % (CI 95 %, 18.6-33.2 %) and 71.1 % (CI 95 %, 62.9-78.1 %), respectively; VD concentrations ≤ 10 ng/mL had 13.3 % (CI 95 %, 8.5-20.2 %) and ≥ 30 ng/mL at 3.7 % (CI 95 %: 1.4-8.6 %). When we contrasted the men to the women, the median concentration of VD did not differ significantly (16 ng/mL vs. 18 ng/mL, p = 0.71). CONCLUSION: In this study, patients with allergic asthma had distinctively reduced VD concentration levels; future research will determine if and how VD affects the severity of asthma.

Antecedentes: La hipovitaminosis D ha sido asociada con diversas enfermedades crónicas como infecciones, enfermedades autoinmunes, enfermedad pulmonar obstructiva crónica, cáncer y asma. Objetivo: Determinar la prevalencia de insuficiencia y deficiencia de vitamina (VD) en adultos con asma alérgica. Métodos: Estudio transversal en el que se analizaron los datos de 135 pacientes. La concentración de VD fue categorizada en suficiente (≥ 30 ng/mL), insuficiente (21-29 ng/mL) y deficiente (≤ 20 ng/mL). La concentración de VD se midió por quimioluminiscencia. Se estimaron las prevalencias de las alteraciones de la VD y sus respectivos intervalos de confianza a 95 % (IC 95 %). Resultados: En la población analizada, 99 fueron mujeres (73.3 %), con edad media de 34.5 ± 10.3 años. La concentración media de VD fue 17.9 ± 6.9 ng/mL (mediana de 17 ng/mL). La prevalencia de insuficiencia y deficiencia de VD fue de 25.2 % (IC 95 %, 18.6-33.2 %) y 71.1 % (IC 95 %, 62.9-78.1 %), respectivamente; las concentraciones de VD ≤ 10 ng/mL representaron 13.3 % (IC 95 %, 8.5-20.2 %) y ≥ 30 ng/mL, 3.7 % (IC 95 %, 1.4-8.6 %). Al contrastar hombres y mujeres, la concentración mediana de VD no difirió significativamente (16 ng/mL versus 18 ng/mL, p = 0.71). Conclusiones: En este estudio, los pacientes con asma alérgica tuvieron concentraciones de VD notoriamente disminuidas. Con futuras investigaciones se podrá evaluar el papel de la VD en la gravedad del asma.

High-affinity RNA binding by a hyperthermophilic single-stranded DNA-binding protein.

Single-stranded DNA-binding proteins (SSBs), including replication protein A (RPA) in eukaryotes, play a central role in DNA replication, recombination, and repair. SSBs utilise an oligonucleotide/oligosaccharide-binding (OB) fold domain to bind DNA, and typically oligomerise in solution to bring multiple OB fold domains together in the functional SSB. SSBs from hyperthermophilic crenarchaea, such as Sulfolobus solfataricus, have an unusual structure with a single OB fold coupled to a flexible C-terminal tail. The OB fold resembles those in RPA, whilst the tail is reminiscent of bacterial SSBs and mediates interaction with other proteins. One paradigm in the field is that SSBs bind specifically to ssDNA and much less strongly to RNA, ensuring that their functions are restricted to DNA metabolism. Here, we use a combination of biochemical and biophysical approaches to demonstrate that the binding properties of S. solfataricus SSB are essentially identical for ssDNA and ssRNA. These features may represent an adaptation to a hyperthermophilic lifestyle, where DNA and RNA damage is a more frequent event.

Binding dynamics of a monomeric SSB protein to DNA: a single-molecule multi-process approach.

Single-stranded DNA binding proteins (SSBs) are ubiquitous across all organisms and are characterized by the presence of an OB (oligonucleotide/oligosaccharide/oligopeptide) binding motif to recognize single-stranded DNA (ssDNA). Despite their critical role in genome maintenance, our knowledge about SSB function is limited to proteins containing multiple OB-domains and little is known about single OB-folds interacting with ssDNA. Sulfolobus solfataricus SSB (SsoSSB) contains a single OB-fold and being the simplest representative of the SSB-family may serve as a model to understand fundamental aspects of SSB:DNA interactions. Here, we introduce a novel approach based on the competition between Förster resonance energy transfer (FRET), protein-induced fluorescence enhancement (PIFE) and quenching to dissect SsoSSB binding dynamics at single-monomer resolution. We demonstrate that SsoSSB follows a monomer-by-monomer binding mechanism that involves a positive-cooperativity component between adjacent monomers. We found that SsoSSB dynamic behaviour is closer to that of Replication Protein A than to Escherichia coli SSB; a feature that might be inherited from the structural analogies of their DNA-binding domains. We hypothesize that SsoSSB has developed a balance between high-density binding and a highly dynamic interaction with ssDNA to ensure efficient protection of the genome but still allow access to ssDNA during vital cellular processes.

A STD-NMR study of the interaction of the Anabaena ferredoxin-NADP+ reductase with the coenzyme.

Ferredoxin-NADP+ reductase (FNR) catalyzes the electron transfer from ferredoxin to NADP+ via its flavin FAD cofactor. To get further insights in the architecture of the transient complexes produced during the hydride transfer event between the enzyme and the NADP+ coenzyme we have applied NMR spectroscopy using Saturation Transfer Difference (STD) techniques to analyze the interaction between FNRox and the oxidized state of its NADP+ coenzyme. We have found that STD NMR, together with the use of selected mutations on FNR and of the non-FNR reacting coenzyme analogue NAD+, are appropriate tools to provide further information about the the interaction epitope.

The transient catalytically competent coenzyme allocation into the active site of Anabaena ferredoxin NADP+ -reductase.

Ferredoxin-NADP(+) reductase (FNR) catalyses the electron transfer from ferredoxin to NADP(+) via its flavin FAD cofactor. A molecular dynamics theoretical approach is applied here to visualise the transient catalytically competent interaction of Anabaena FNR with its coenzyme, NADP(+). The particular role of some of the residues identified as key in binding and accommodating the 2'P-AMP moiety of the coenzyme is confirmed in molecular terms. Simulations also indicate that the architecture of the active site precisely contributes to the orientation of the N5 of the FAD isoalloxazine ring and the C4 of the coenzyme nicotinamide ring in the conformation of the catalytically competent hydride transfer complex and, therefore, contributes to the efficiency of the process. In particular, the side chain of the C-terminal Y303 in Anabaena FNR appears key to providing the optimum geometry by reducing the stacking probability between the isoalloxazine and nicotinamide rings, thus providing the required co-linearity and distance among the N5 of the flavin cofactor, the C4 of the coenzyme nicotinamide and the hydride that has to be transferred between them. All these factors are highly related to the reaction efficiency, mechanism and reversibility of the process.

Role of specific residues in coenzyme binding, charge-transfer complex formation, and catalysis in Anabaena ferredoxin NADP+-reductase.

Two transient charge-transfer complexes (CTC) form prior and upon hydride transfer (HT) in the reversible reaction of the FAD-dependent ferredoxin-NADP+ reductase (FNR) with NADP+/H, FNR(ox)-NADPH (CTC-1), and FNR(rd)-NADP+ (CTC-2). Spectral properties of both CTCs, as well as the corresponding interconversion HT rates, are here reported for several Anabaena FNR site-directed mutants. The need for an adequate initial interaction between the 2'P-AMP portion of NADP+/H and FNR that provides subsequent conformational changes leading to CTC formation is further confirmed. Stronger interactions between the isoalloxazine and nicotinamide rings might relate with faster HT processes, but exceptions are found upon distortion of the active centre. Thus, within the analyzed FNR variants, there is no strict correlation between the stability of the transient CTCs formation and the rate of the subsequent HT. Kinetic isotope effects suggest that, while in the WT, vibrational enhanced modulation of the active site contributes to the tunnel probability of HT; complexes of some of the active site mutants with the coenzyme hardly allow the relative movement of isoalloxazine and nicotinamide rings along the HT reaction. The architecture of the WT FNR active site precisely contributes to reduce the stacking probability between the isoalloxazine and nicotinamide rings in the catalytically competent complex, modulating the angle and distance between the N5 of the FAD isoalloxazine and the C4 of the coenzyme nicotinamide to values that ensure efficient HT processes.

Mechanism of the hydride transfer between Anabaena Tyr303Ser FNR(rd)/FNR(ox) and NADP+/H. A combined pre-steady-state kinetic/ensemble-averaged transition-state theory with multidimensional tunneling study.

The flavoenzyme ferredoxin-NADP(+) reductase (FNR) catalyzes the production of NADPH during photosynthesis. The hydride-transfer reactions between the Anabaena mutant Tyr303Ser FNR(rd)/FNR(ox) and NADP(+)/H have been studied both experimentally and theoretically. Stopped-flow pre-steady-state kinetic measurements have shown that, in contrast to that observed for WT FNR, the physiological hydride transfer from Tyr303Ser FNR(rd) to NADP(+) does not occur. Conversely, the reverse reaction does take place with a rate constant just slightly slower than for WT FNR. This latter process shows temperature-dependent rates, but essentially temperature independent kinetic isotope effects, suggesting the reaction takes place following the vibration-driven tunneling model. In turn, ensemble-averaged variational transition-state theory with multidimensional tunneling calculations provide reaction rate constant values and kinetic isotope effects that agree with the experimental results, the experimental and the theoretical values for the reverse process being noticeably similar. The reaction mechanism behind these hydride transfers has been analyzed. The formation of a close contact ionic pair FADH(-):NADP(+) surrounded by the polar environment of the enzyme in the reactant complex of the mutant might be the cause of the huge difference between the direct and the reverse reaction.

Protein motifs involved in coenzyme interaction and enzymatic efficiency in anabaena ferredoxin-NADP+ reductase.

Ferredoxin-NADP+ reductases (FNRs) must determine the coenzyme specificity and allow the transient encounter between N5 of its flavin cofactor and C4 of the coenzyme nicotinamide for efficient hydride transfer. Combined site-directed replacements in different putative determinants of the FNR coenzyme specificity were simultaneously produced. The resulting variants were structurally and functionally analyzed for their binding and hydride transfer abilities to the FNR physiological coenzyme NADP+/H, as well as to NAD+/H. The previously studied Y303S mutation is the only one that significantly enhances specificity for NAD+. Combination of mutations from the pyrophosphate or 2'-phosphate regions, even including Y303S, does not improve activity with NAD+, despite structures of these FNRs show how particular coenzyme-binding regions resembled motifs found in NAD+/H-dependent enzymes of the FNR family. Therefore, the "rational approach" did not succeed well, and coenzyme specificity redesign in the FNR family will be more complex than that anticipated in other NADP+/NAD+ families.

Flavodoxin: a compromise between efficiency and versatility in the electron transfer from Photosystem I to Ferredoxin-NADP(+) reductase.

Under iron-deficient conditions Flavodoxin (Fld) replaces Ferredoxin in Anabaena as electron carrier from Photosystem I (PSI) to Ferredoxin-NADP(+) reductase (FNR). Several residues modulate the Fld interaction with FNR and PSI, but no one appears as specifically critical for efficient electron transfer (ET). Fld shows a strong dipole moment, with its negative end directed towards the flavin ring. The role of this dipole moment in the processes of interaction and ET with positively charged surfaces exhibited by PSI and FNR has been analysed by introducing single and multiple charge reversal mutations on the Fld surface. Our data confirm that in this system interactions do not rely on a precise complementary surface of the reacting molecules. In fact, they indicate that the initial orientation driven by the alignment of dipole moment of the Fld molecule with that of the partner contributes to the formation of a bunch of alternative binding modes competent for the efficient ET reaction. Additionally, the fact that Fld uses different interaction surfaces to dock to PSI and to FNR is confirmed.

Flavodoxin-mediated electron transfer from photosystem I to ferredoxin-NADP+ reductase in Anabaena: role of flavodoxin hydrophobic residues in protein-protein interactions.

Three surface hydrophobic residues located at the Anabaena flavodoxin (Fld) putative complex interface with its redox partners were replaced by site-directed mutagenesis. The effects of these replacements on Fld interaction with both its physiological electron donor, photosystem I (PSI), and its electron acceptor, ferredoxin-NADP+ reductase (FNR), were analyzed. Trp57, Ile59, and Ile92 contributed to the optimal orientation and tightening of the FNR:Fld and PSI:Fld complexes. However, these side chains did not appear to be involved in crucial specific interactions, but rather contributed to the obtainment of the optimal orientation and distance of the redox centers required for efficient electron transfer. This supports the idea that the interaction of Fld with its partners is less specific than that of ferredoxin and that more than one orientation is efficient for electron transfer in these transient complexes. Additionally, for some of the analyzed processes, WT Fld seems not to be the most optimized molecular species. Therefore, subtle changes at the isoalloxazine environment not only influence the Fld binding abilities, but also modulate the electron exchange processes by producing different orientations and distances between the redox centers. Finally, the weaker apoflavodoxin interaction with FNR suggests that the solvent-accessible region of FMN plays a role either in complex formation with FNR or in providing the adequate conformation of the FNR binding region in Fld.

Tuning of the FMN binding and oxido-reduction properties by neighboring side chains in Anabaena flavodoxin.

Contribution of three regions (phosphate-binding, 50's and 90's loops) of Anabaena apoflavodoxin to FMN binding and reduction potential was studied. Thr12 and Glu16 did not influence FMN redox properties, but Thr12 played a role in FMN binding. Replacement of Trp57 with Glu, Lys or Arg moderately shifted E(ox/sq) and E(sq/hq) and altered the energetic of the FMN redox states binding profile. Our data indicate that the side chain of position 57 does not modulate E(ox/sq) by aromatic stacking or solvent exclusion, but rather by influencing the relative strength of the H-bond between the N(5) of the flavin and the Asn58-Ile59 bond. A correlation was observed between the isoalloxazine increase in solvent accessibility and less negative E(sq/hq). Moreover, E(sq/hq) became less negative as positively charged residues were added near to the isoalloxazine. Ile59 and Ile92 were simultaneously mutated to Ala or Glu. These mutations impaired FMN binding, while shifting E(sq/hq) to less negative values and E(ox/sq) to more negative. These effects are discussed on the bases of the X-ray structures of some of the Fld mutants, suggesting that in Anabaena Fld the structural control of both electron transfer steps is much more subtle than in other Flds.

Catalytic mechanism of hydride transfer between NADP+/H and ferredoxin-NADP+ reductase from Anabaena PCC 7119.

The mechanism of hydride transfer between Anabaena FNR and NADP+/H was analysed using for the first time stopped-flow photodiode array detection and global analysis deconvolution. The results indicated that the initial spectral changes, occurring within the instrumental dead time upon reaction of FNR with NADP+/H, included not only the initial interaction and complex formation, but also the first subsequent steps of the sequential reactions that involve hydride transfer. Two different charge-transfer complexes formed prior and upon hydride transfer, FNRox-NADPH and FNRrd-NADP+. Detectable amounts of FNRox-NADPH were found at equilibrium, but FNRrd-NADP+ accumulated to a small extent and quickly evolved. The spectral properties of both charge-transfer complexes, for the first time in Anabaena FNR, as well as the corresponding inter-conversion hydride transfer rates were obtained. The need of an adequate initial interaction between NADP+/H and FNR, and subsequent conformational changes, was also established by studying the reactions of two FNR mutants.

Exact analysis of heterotropic interactions in proteins: Characterization of cooperative ligand binding by isothermal titration calorimetry.

Intramolecular interaction networks in proteins are responsible for heterotropic ligand binding cooperativity, a biologically important, widespread phenomenon in nature (e.g., signaling transduction cascades, enzymatic cofactors, enzymatic allosteric activators or inhibitors, gene transcription, or repression). The cooperative binding of two (or more) different ligands to a macromolecule is the underlying principle. To date, heterotropic effects have been studied mainly kinetically in enzymatic systems. Until now, approximate approaches have been employed for studying equilibrium heterotropic ligand binding effects, except in two special cases in which an exact analysis was developed: independent binding (no cooperativity) and competitive binding (maximal negative cooperativity). The exact analysis and methodology for characterizing ligand binding cooperativity interactions in the general case (any degree of cooperativity) using isothermal titration calorimetry are presented in this work. Intramolecular interaction pathways within the allosteric macromolecule can be identified and characterized using this methodology. As an example, the thermodynamic characterization of the binding interaction between ferredoxin-NADP+ reductase and its three substrates, NADP+, ferredoxin, and flavodoxin, as well as the characterization of their binding cooperativity interaction, is presented.

Towards a new interaction enzyme:coenzyme.

Ferredoxin-NADP(+) reductase catalyses NADP(+) reduction, being specific for NADP(+)/H. To understand coenzyme specificity determinants and coenzyme specificity reversion, mutations at the NADP(+)/H pyrophosphate binding and of the C-terminal regions have been simultaneously introduced in Anabaena FNR. The T155G/A160T/L263P/Y303S mutant was produced. The mutated enzyme presents similar k(cat) values for NADPH and NADH, around 2.5 times slower than that reported for WT FNR with NADPH. Its K(m) value for NADH decreased 20-fold with regard to WT FNR, whereas the K(m) for NADPH remains similar. The combined effect is a much higher catalytic efficiency for NAD(+)/H, with a minor decrease of that for NADP(+)/H. In the mutated enzyme, the specificity for NADPH versus NADH has been decreased from 67,500 times to only 12 times, being unable to discriminate between both coenzymes. Additionally, giving the role stated for the C-terminal Tyr in FNR, its role in the energetics of the FAD binding has been analysed.

Anabaena flavodoxin as an electron carrier from photosystem I to ferredoxin-NADP+ reductase. Role of flavodoxin residues in protein-protein interaction and electron transfer.

Biochemical and structural studies indicate that electrostatic and hydrophobic interactions are critical in the formation of optimal complexes for efficient electron transfer (ET) between ferredoxin-NADP(+) reductase (FNR) and ferredoxin (Fd). Moreover, it has been shown that several charged and hydrophobic residues on the FNR surface are also critical for the interaction with flavodoxin (Fld), although, so far, no key residue on the Fld surface has been found to be the counterpart of such FNR side chains. In this study, negatively charged side chains on the Fld surface have been individually modified, either by the introduction of positive charges or by their neutralization. Our results indicate that although Glu16, Glu20, Glu61, Asp65, and Asp96 contribute to the orientation and optimization of the Fld interaction, either with FNR or with photosystem I (PSI) (presumably through the formation of salt bridges), for efficient ET, none of these side chains is involved in the formation of crucial salt bridges for optimal interaction with FNR. These data support the idea that the FNR-Fld interaction is less specific than the FNR-Fd interaction. However, analysis of the reactivity of these mutated Flds toward the membrane-anchored PSI complex indicated that all mutants, except Glu16Gln, lack the ability to form a stable complex with PSI. Thr12, Thr56, Asn58, and Asn97 are present in the close environment of the isoalloxazine ring of FMN in Anabaena Fld. Their roles in the interaction with and ET to FNR and PSI have also been studied. Mutants at these Fld positions indicate that residues in the close environment of the isoalloxazine ring modulate the ability of Fld to bind to and to exchange electrons with its physiological counterparts.