Functional plasticity of Capsicum annuum var. glabriusculum through multiple traits.

The diversity of functional traits still has not been studied enough in model plant species, even less so in little-known species. This experiment was carried out under the extreme heat of Sonoran Desert, using shading nets and under conditions where the availability of water and nutrients was not a stress factor. We evaluated how the low, intermediate and high sunlight regimes impact survival and promote multiple alterations on phenological and ecophysiological response of cultivated Capsicum annuum var. glabriusculum plants. Extremely warm temperatures promoted a high heat sum in degrees days throughout plants development. Most plants grown in high sunlight regimes did not survive; under intermediate sunlight regimes survival was high and plants developed vegetative and reproductively; but under low sunlight regimes plants survival was high; however, they developed just vegetatively. Photosynthetic response to light suggests that plants are physiologically acclimated to low and intermediate irradiance, whereas the CO2 assimilation curves suggest contrasting photosynthetic capacity traits. Under the intermediate sunlight regimes, plants strengthened their performance through multiple functional traits (e.g. CO2 and water diffusion traits, photosynthetic capacity, respiration, among others). Consequently, their biomass gain was faster and proportionally higher by 76 % with an investment of 14 % in fruits development. The principal components analysis extracted the main explanatory functional traits: photosynthetic nitrogen allocation, stomatal limitation, mesophyll conductance, Rubisco maximum carboxylation velocity, among others. In conclusion, phenological response and multiple functional traits determine plants acclimation to sunlight regimes and extremely warm temperatures in short term.

Neutropenic Enterocolitis: Case report and literature review

Typhlitis, is also known as neutropenic enterocolitis, affects the cecum and distal ileum. It was frequently encountered in pediatric patients who were undergoing treatment for leukemia. Nonetheless, it can affect adult patients, regardless of the cause of the immunosuppression. We report the case of a 20-year-old patient who was receiving chemotherapy for Osteosarcoma, who had a 6-day history of nausea and vomiting, fever sensation, diarrhea, and diffuse abdominal pain. Physical examination was relevant for hemodynamic instability, a distended and tender abdomen predominantly in the right iliac fossa. The laboratory workup showed severe neutropenia, thrombocytopenia, and electrolyte disturbances. The image studies evidenced edema of the ascending colon and cecum. Treatment was started with vasopressor support, correction of electrolyte alterations, blood cell and platelet transfusion, G-CSF, hydration, broad spectrum antibiotic therapy, initially with adequate clinical and laboratory response. After a few days, he presented lower gastrointestinal bleeding which was treated by conservative management. In conclusion, typhlitis must be suspected in every patient developing neutropenia as a reaction to chemotherapy and who also presents gastrointestinal symptoms, such as nausea, vomiting, diarrhea, and intense abdominal pain.

La tiflitis o también denominada enterocolitis neutropénica afecta el ciego e íleon distal. Fue descrita comúnmente en pacientes pediátricos sometidos a tratamiento para leucemia; sin embargo, puede afectar al paciente adulto independiente de la causa que origine la inmunosupresión. Presentamos el caso de un paciente de 20 años con antecedente de osteosarcoma, quien acude con tiempo de enfermedad de 6 días caracterizado por náuseas y vómitos, sensación de alza térmica, diarrea y dolor abdominal difuso. Al examen físico se encuentra hemodinámicamente inestable, abdomen distendido y se corrobora el dolor abdominal a predominio en fosa iliaca derecha. Los exámenes de laboratorio mostraron neutropenia severa, trombocitopenia y alteraciones hidroelectrolíticas. En los estudios de imágenes la TC evidenció edema de asas delgadas, así como edema de pared del colon ascendente y ciego. Se inició tratamiento con soporte vasopresor, corrección de alteraciones hidroelectrolíticas, transfusión de paquetes globulares y plaquetas, G-CSF, hidratación, terapia antibiótica de amplio espectro, inicialmente con adecuada respuesta clínica y laboratorial. Al cabo de unos días presentó hemorragia digestiva baja como complicación que fue tratada mediante manejo conservador. En conclusión, la tiflitis debe sospecharse en todo paciente que desarrolla neutropenia luego de quimioterapia y que presenta síntomas gastrointestinales como náuseas, vómitos, diarrea y dolor abdominal intenso. El manejo conservador, en pacientes con hemorragia digestiva y tiflitis, que involucra corrección de coagulopatía, transfusión de paquetes globulares e hidratación, puede ser el adecuado en pacientes que no cursan con inestabilidad hemodinámica y logran controlar el sangrado.

Biosynthesis, Gene Expression, and Pharmacological Properties of Triterpenoids of Ganoderma Species (Agaricomycetes): A Review.

For more than 6 millennia, Ganoderma species have been used in traditional Asian medicine due to their health benefits. Ganoderma synthesizes several compounds with biological activity, including lanostane-type triterpenoids like ganoderic acids (GAs), lucidones, and colosolactones. These triterpenoids have been investigated for their antiviral, hypoglycemic, and anticancer effects. GAs are highly oxygenated triterpenoids with different functional groups attached to lanostane skeleton. Their great chemical diversity makes GAs prospects for the development of new drugs to treat multiple illnesses such as cancer. The effect of GAs against cancer cells has been associated with their capability to inhibit specific targets such as STAT3, to induce apoptosis and cell cycle blockage, and to increase natural killer cell activity. Due to the biological activity of these molecules, novel strategies are being developed for Ganoderma production mainly by liquid cultivation, gene overexpression (HMGR, SQS, LS) by elicitors, and modified growing conditions (carbon and nitrogen sources, pH, temperature), which induce reactive oxygen species production, key compounds for secondary metabolism. In addition, some transcription factors are mainly expressed under stress conditions, such as cytochrome P450 genes, which participate in the regulation of triterpenoid synthesis. The fermentation process has been scaled up to a 300-L bioreactor, which shows good GA production. This article reviews current knowledge on bioactive triterpenoids of Ganoderma and their production, biosynthesis, and pharmacological properties, emphasizing gene expression in liquid culture. It also discusses the lack of information regarding other species with high potential.

Phenolic compounds of Phellinus spp. with antibacterial and antiviral activities.

Phellinus Quél is one of the largest genera of Hymenochaetaceae; it comprises about 220 species widely distributed on Earth. Most Phellinus species are lignicolous mushrooms that accumulate bioactive compounds. This research studied the phenolic composition of Phellinus spp. and their relationship with antibacterial and antiviral capacity. Phenolics were extracted from Phellinus badius, P. fastuosus, and P. grenadensis; their antiviral and antibacterial activities were evaluated against Listeria monocytogenes, Staphylococcus aureus, Salmonella enterica, and Escherichia coli O157: H7; and the bacteriophages MS2 and Φ- × 174. Gallic acid, chlorogenic acid, caffeic acid, epicatechin, ferulic acid, catechin, 1,3-dicaffeoylquinic acid, p-coumaric acid, and rutin were found in different proportions among Phellinus spp. Total phenolic content ranged from 96 to 209 mg GAE/g, and total flavonoids from 10 to 27 QE/g. The minimum inhibitory concentrations of P. badius, P. grenadensis, and P. fastuosus against E. coli O157: H7 were 13, 20, and 27 mg/mL, against S. enterica were 20, 30, and 15 mg/mL, and against L. monocytogenes were 10, 15, and 25 mg/mL, respectively. The phenolic content was better correlated with the antibacterial effect against E. coli O157: H7 and L. monocytogenes (r = 0.8-0.9), but not against S. enterica (r = 0.05). The antiviral activity of the extracts (0.9 mg/mL) was 29 to 41% against MS2 and 27 to 38% for Φ-X174 virus (r = 0.8-0.9). In silico analysis showed binding energy values of - 7.9 and - 4.8 kcal/mol between the identified phenolic compounds and the M and G proteins of each virus. The antibacterial and antiviral properties of Phellinus species were correlated with the phenolic content.

Neutropenic Enterocolitis: Case report and literature review.

Typhlitis, is also known as neutropenic enterocolitis, affects the cecum and distal ileum. It was frequently encountered in pediatric patients who were undergoing treatment for leukemia. Nonetheless, it can affect adult patients, regardless of the cause of the immunosuppression. We report the case of a 20-year-old patient who was receiving chemotherapy for Osteosarcoma, who had a 6-day history of nausea and vomiting, fever sensation, diarrhea, and diffuse abdominal pain. Physical examination was relevant for hemodynamic instability, a distended and tender abdomen predominantly in the right iliac fossa. The laboratory workup showed severe neutropenia, thrombocytopenia, and electrolyte disturbances. The image studies evidenced edema of the ascending colon and cecum. Treatment was started with vasopressor support, correction of electrolyte alterations, blood cell and platelet transfusion, G-CSF, hydration, broad spectrum antibiotic therapy, initially with adequate clinical and laboratory response. After a few days, he presented lower gastrointestinal bleeding which was treated by conservative management. In conclusion, typhlitis must be suspected in every patient developing neutropenia as a reaction to chemotherapy and who also presents gastrointestinal symptoms, such as nausea, vomiting, diarrhea, and intense abdominal pain.

Negative Cooperativity in NAD(P)H Quinone Oxidoreductase 1 (NQO1).

NAD(P)H quinone oxidoreductase-1 (NQO1) is a homodimeric protein that acts as a detoxifying enzyme or as a chaperone protein. Dicourmarol interacts with NQO1 at the NAD(P)H binding site and can both inhibit enzyme activity and modulate the interaction of NQO1 with other proteins. We show that the binding of dicoumarol and related compounds to NQO1 generates negative cooperativity between the monomers. This does not occur in the presence of the reducing cofactor, NAD(P)H, alone. Alteration of Gly150 (but not Gly149 or Gly174) abolished the dicoumarol-induced negative cooperativity. Analysis of the dynamics of NQO1 with the Gaussian network model indicates a high degree of collective motion by monomers and domains within NQO1. Ligand binding is predicted to alter NQO1 dynamics both proximal to the ligand binding site and remotely, close to the second binding site. Thus, drug-induced modulation of protein motion might contribute to the biological effects of putative inhibitors of NQO1.

Review of Bioactive Molecules Production, Biomass, and Basidiomata of Shiitake Culinary-Medicinal Mushrooms, Lentinus edodes (Agaricomycetes).

Lentinus edodes (shiitake) is a basidiomycete that has been consumed for more than 2000 years because of its nutritional value and health benefits. It has a low lipid content, high fiber content, and a considerable amount of proteins; it also contains B vitamins and minerals in addition to a wide range of functional metabolites including polysaccharides, polysaccharopeptides, lectins, and secondary metabolites with bioactivity, e.g., lentinan, a ß-(1-3)-glucan with immunomodulatory activity, among others. Extracts and pure compounds of shiitake exhibit antibacterial, antifungal, cytostatic, antioxidant, anticancer, and immunomodulatory activity. Because of these attributes, different products derived from shiitake are on the market and are sold as dietary supplements. The traditional substrate for shiitake production is oak wood, yet the search for unconventional substrates has intensified over the past three decades. In particular, submerged cultivation of medicinal mushrooms has attracted great interest because it enables greater control of different fermentation factors to obtain products of interest. However, it is necessary to perform in vivo studies to determine the appropriate doses, side effects, and action spectrum of different bioactive compounds and fractions as well as to improve their production in liquid media and to potentiate their activity. We present an updated review of existing studies on the production of biomass and bioactive compounds of L. edodes in liquid culture and on solid fermentation for obtaining secondary mycelia and basidiomata.

Vineyard Pruning Waste Improves Bioconversion and Chemical Composition of Native Ganoderma spp. (Agaricomycetes) Strains from Mexico.

The aim of this work was to evaluate wood chips from vineyard prunings (VPs) as a potential substrate in cultures of Ganoderma spp. Biological efficiency (BE), production rate (PR), yield, and protein and fat contents of basidiomes increased when the wild strains G. oerstedii and G. subincrustatum, which were isolated from the Sonoran Desert, were cultivated on VPs. The mineral content of the basidiomes varied depending on the strain and substrate. The carbohydrate and phenol contents of the different substrate combinations and of the basidiomes were similar among strains. However, the carbohydrate and phenol contents of the substrates did not correlate with an increase in BE. Conversely, the high availability of protein, fat, and hemicellulose in VPs positively correlated with increases in BE, in yield, and in the protein and fat contents of the basidiomes of the wild strains. Our results clearly demonstrate that, in comparison with the traditional substrate of oak wood, VPs improve the production and biochemical composition of basidiomes. Therefore, cultivation of Ganoderma spp. on VPs is an environmentally friendly strategy for increasing their nutritional value and for cultivating these mushrooms for other biotechnological applications.

Physiological response of Cucurbita pepo var. pepo mycorrhized by Sonoran desert native arbuscular fungi to drought and salinity stresses

ABSTRACT Plants response to symbiosis with arbuscular mycorrhizal fungi (AMF) under water stress is important to agriculture. Under abiotic stress conditions native fungi are more effective than exotics in improving plant growth and water status. Mycorrhization efficiency is related to soil fungi development and energy cost-benefit ratio. In this study, we assessed the effect on growth, water status and energy metabolism of Cucurbita pepo var. pepo when inoculated with native AMF from the Sonoran desert Mexico (mixed isolate and field consortium), and compared with an exotic species from a temperate region, under drought, low and high salinity conditions. Dry weights, leaf water content, water and osmotic potentials, construction costs, photochemistry and mycorrhization features were quantified. Under drought and low salinity conditions, the mixed isolate increased plant growth and leaf water content. Leaf water potential was increased only by the field consortium under drought conditions (0.5-0.9 MPa). Under high salinity, the field consortium increased aerial dry weight (more than 1 g) and osmotic potential (0.54 MPa), as compared to non-mycorrhized controls. Plants inoculated with native AMF, which supposedly diminish the effects of stress, exhibited low construction costs, increased photochemical capacity, and grew larger external mycelia in comparison to the exotic inoculum.

Physiological response of Cucurbita pepo var. pepo mycorrhized by Sonoran desert native arbuscular fungi to drought and salinity stresses.

Plants response to symbiosis with arbuscular mycorrhizal fungi (AMF) under water stress is important to agriculture. Under abiotic stress conditions native fungi are more effective than exotics in improving plant growth and water status. Mycorrhization efficiency is related to soil fungi development and energy cost-benefit ratio. In this study, we assessed the effect on growth, water status and energy metabolism of Cucurbita pepo var. pepo when inoculated with native AMF from the Sonoran desert Mexico (mixed isolate and field consortium), and compared with an exotic species from a temperate region, under drought, low and high salinity conditions. Dry weights, leaf water content, water and osmotic potentials, construction costs, photochemistry and mycorrhization features were quantified. Under drought and low salinity conditions, the mixed isolate increased plant growth and leaf water content. Leaf water potential was increased only by the field consortium under drought conditions (0.5-0.9MPa). Under high salinity, the field consortium increased aerial dry weight (more than 1g) and osmotic potential (0.54MPa), as compared to non-mycorrhized controls. Plants inoculated with native AMF, which supposedly diminish the effects of stress, exhibited low construction costs, increased photochemical capacity, and grew larger external mycelia in comparison to the exotic inoculum.

Transglutaminase-2 interaction with heparin: identification of a heparin binding site that regulates cell adhesion to fibronectin-transglutaminase-2 matrix.

Heparan sulfate proteoglycans are critical binding partners for extracellular tranglutaminase-2 (TG2), a multifunctional protein involved in tissue remodeling events related to organ fibrosis and cancer progression. We previously showed that TG2 has a strong affinity for heparan sulfate (HS)/heparin and reported that the heparan sulfate proteoglycan syndecan-4 acts as a receptor for TG2 via its HS chains in two ways: by increasing TG2-cell surface trafficking/externalization and by mediating RGD-independent cell adhesion to fibronectin-TG2 matrix during wound healing. Here we have investigated the molecular basis of this interaction. Site-directed mutagenesis revealed that either mutation of basic RRWK (262-265) or KQKRK (598-602) clusters, forming accessible heparin binding sequences on the TG2 three-dimensional structure, led to an almost complete reduction of heparin binding, indicating that both clusters contribute to form a single binding surface. Mutation of residues Arg(19) and Arg(28) also led to a significant reduction in heparin binding, suggesting their involvement. Our findings indicate that the heparin binding sites on TG2 mainly comprise two clusters of basic amino acids, which are distant in the linear sequence but brought into spatial proximity in the folded "closed" protein, forming a high affinity heparin binding site. Molecular modeling showed that the identified site can make contact with a single heparin-derived pentasaccharide. The TG2-heparin binding mutants supported only weak RGD-independent cell adhesion compared with wild type TG2 or mutants with retained heparin binding, and both heparin binding clusters were critical for TG2-mediated cell adhesion. These findings significantly advance our knowledge of how HS/heparin influences the adhesive function of TG2.

Quantitative changes in the biochemical composition of lignocellulosic residues during the vegetative growth of Lentinula edodes

The chemical changes in barley-straw (BS), wheat-straw (WS) and vineyard-pruning (VP) substrates were determined during colonization of Lentinula edodes mycelia (during primordium development) in solid state fermentation. Primordia appeared 39-50 days after inoculation. VP appeared to promote early sporophore initiation. The concentration of hemicellulose in BS and VP decreased gradually from 25.5 percent to 15.6 percent and from 15.8 percent to 12.3 percent, respectively. However in WS, hemicellulose decreased from 27.2 percent to 9.5 percent. Lignin broke down continuously in BS and WS, with 31.8 percent and 34.4 percent degradation, respectively; higher than that of cellulose. During the pinning stage, the C:N ratio decreased in VP and BS, but not in WS. On all substrates the phenols decreased notably throughout the first week of mycelial growth. The time elapsed (days) to pinning was positively correlated with cellulose content (r=0.89), total sugar (r=0.85) and inversely correlated to lignin (r=-1.00) and phenol content (r=-0.55).

Quantitative changes in the biochemical composition of lignocellulosic residues during the vegetative growth of Lentinula edodes.

The chemical changes in barley-straw (BS), wheat-straw (WS) and vineyard-pruning (VP) substrates were determined during colonization of Lentinula edodes mycelia (during primordium development) in solid state fermentation. Primordia appeared 39-50 days after inoculation. VP appeared to promote early sporophore initiation. The concentration of hemicellulose in BS and VP decreased gradually from 25.5% to 15.6% and from 15.8% to 12.3%, respectively. However in WS, hemicellulose decreased from 27.2% to 9.5%. Lignin broke down continuously in BS and WS, with 31.8% and 34.4% degradation, respectively; higher than that of cellulose. During the pinning stage, the C:N ratio decreased in VP and BS, but not in WS. On all substrates the phenols decreased notably throughout the first week of mycelial growth. The time elapsed (days) to pinning was positively correlated with cellulose content (r=0.89), total sugar (r=0.85) and inversely correlated to lignin (r=-1.00) and phenol content (r=-0.55).

Domain motion in cytochrome P450 reductase: conformational equilibria revealed by NMR and small-angle x-ray scattering.

NADPH-cytochrome P450 reductase (CPR), a diflavin reductase, plays a key role in the mammalian P450 mono-oxygenase system. In its crystal structure, the two flavins are close together, positioned for interflavin electron transfer but not for electron transfer to cytochrome P450. A number of lines of evidence suggest that domain motion is important in the action of the enzyme. We report NMR and small-angle x-ray scattering experiments addressing directly the question of domain organization in human CPR. Comparison of the (1)H-(15)N heteronuclear single quantum correlation spectrum of CPR with that of the isolated FMN domain permitted identification of residues in the FMN domain whose environment differs in the two situations. These include several residues that are solvent-exposed in the CPR crystal structure, indicating the existence of a second conformation in which the FMN domain is involved in a different interdomain interface. Small-angle x-ray scattering experiments showed that oxidized and NADPH-reduced CPRs have different overall shapes. The scattering curve of the reduced enzyme can be adequately explained by the crystal structure, whereas analysis of the data for the oxidized enzyme indicates that it exists as a mixture of approximately equal amounts of two conformations, one consistent with the crystal structure and one a more extended structure consistent with that inferred from the NMR data. The correlation between the effects of adenosine 2',5'-bisphosphate and NADPH on the scattering curve and their effects on the rate of interflavin electron transfer suggests that this conformational equilibrium is physiologically relevant.

Effects on goat milk quality of the presence of Mycoplasma spp. in herds without symptoms of contagious agalactia.

This study was designed to assess the possible effects of mycoplasmas on the quality of milk produced by goat herds in a contagious agalactia (CA) endemic area with absence of classical symptoms. Several factors related to milk quality (percentages of fat, total protein, lactose and total solids, standard plate counts (SPC) and presence of Staphylococcus aureus) were compared in mycoplasma-infected and non-infected herds. To define the CA status of 26 herds on the island of Lanzarote (Spain), where CA is endemic, 570 individual milk samples and 266 bulk tank milk (BTM) samples were microbiologically analysed for the presence of Mycoplasma spp. A herd was considered infected by mycoplasmas when at least a sample (individual or BTM) was positive. BTM samples were also used to determine milk quality parameters. Mycoplasma infection was confirmed in 13 herds. A total of 31, 10 and 11 strains of Mycoplasma mycoides subsp. mycoides LC (MmmLC), Mp. agalactiae and Mp. capricolum subsp. capricolum were isolated. No significant differences were observed between the least square means of the variables fat, total protein, lactose and total solids or SPC recorded for the infected v. non-infected herds. The Staph. aureus status of a herd was also found to be independent of the presence of Mycoplasma spp. Our findings indicate that neither the presence of mycoplasmas in a goat herd with absence of classical symptoms seem to compromise the quality of the BTM.

Conformational dynamics and the energetics of protein--ligand interactions: role of interdomain loop in human cytochrome P450 reductase.

A combination of mutagenesis, calorimetry, kinetics, and small-angle X-ray scattering (SAXS) has been used to study the mechanism of ligand binding energy propagation through human cytochrome P450 reductase (CPR). Remarkably, the energetics of 2',5'-ADP binding to R597 at the FAD-binding domain are affected by mutations taking place at an interdomain loop located 60 A away. Either deletion of a 7 amino acid long segment (T236-G237-E238-E239-S240-S241-I242) or its replacement by poly-proline repeats (5 and 10 residues) results in a significant increase in 2',5'-ADP enthalpy of binding (DeltaHB). This is accompanied by a decrease in the number of thermodynamic microstates available for the ligand-CPR complex. Moreover, the estimated heat capacity change (DeltaCp) for this interaction changes from -220 cal mol-1 K-1 in the wild-type enzyme to -580 cal mol-1 K-1 in the deletion mutant. Pre-steady-state kinetics measurements reveal a 50-fold decrease in the microscopic rate for interdomain (FAD --> FMN) electron transfer in the deletion mutant (kobs = 0.4 s-1). Multiple turnover cytochome c reduction assays indicate that these mutations impair the ability of the FMN-binding domain to shuttle electrons from the FAD-binding domain to the cytochrome partner. Binding of 2',5'-ADP to wild-type CPR triggers a large-scale structural rearrangement resulting in the complex having a more compact domain organization, and the maximum molecular dimension (Dmax) decreases from 110 A in ligand-free enzyme to 100 A in the ligand-bound CPR. The SAXS experiments also demonstrate that what is affected by the mutations is indeed the relative diffusional motion of the domains. Furthemore, ab initio shape reconstruction and homology modeling would suggest that-in the deletion mutant-hindering of domain motion occurs concomitantly with dimerization. The results presented here show that the energetics of this highly localized interaction (2',5'-ADP binding) have a global character, and are highly sensitive to functional structural dynamics involving distal domains. These findings support early theoretical studies which postulate a single protein molecule to be a real, independent thermodynamic ensemble.

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.

Global effects of the energetics of coenzyme binding: NADPH controls the protein interaction properties of human cytochrome P450 reductase.

The thermodynamics of coenzyme binding to human cytochrome P450 reductase (CPR) and its isolated FAD-binding domain have been studied by isothermal titration calorimetry. Binding of 2',5'-ADP, NADP(+), and H(4)NADP, an isosteric NADPH analogue, is described in terms of the dissociation binding constant (K(d)), the enthalpy (DeltaH(B)) and entropy (TDeltaS(B)) of binding, and the heat capacity change (DeltaC(p)). This systematic approach allowed the effect of coenzyme redox state on binding to CPR to be determined. The recognition and stability of the coenzyme-CPR complex are largely determined by interaction with the adenosine moiety (K(d2)(')(,5)(')(-ADP) = 76 nM), regardless of the redox state of the nicotinamide moiety. Similar heat capacity change (DeltaC(p)) values for 2',5'-ADP (-210 cal mol(-)(1) K(-)(1)), NADP(+) (-230 cal mol(-)(1) K(-)(1)), and H(4)NADP (-220 cal mol(-)(1) K(-)(1)) indicate no significant contribution from the nicotinamide moiety to the binding interaction surface. The coenzyme binding stoichiometry to CPR is 1:1. This result validates a recently proposed one-site kinetic model [Daff, S. (2004) Biochemistry 43, 3929-3932] as opposed to a two-site model previously suggested by us [Gutierrez, A., Lian, L.-Y., Wolf, C. R., Scrutton, N. S., and Roberts, C. G. K. (2001) Biochemistry 40, 1964-1975]. Calorimetric studies in which binding of 2',5'-ADP to CPR (TDeltaS(B) = -13400 +/- 200 cal mol(-)(1), 35 degrees C) was compared with binding of the same ligand to the isolated FAD-binding domain (TDeltaS(B) = -11200 +/- 300 cal mol(-)(1), 35 degrees C) indicate that the number of accessible conformational substates of the protein increases upon 2',5'-ADP binding in the presence of the FMN-binding domain. This pattern was consistently observed along the temperature range that was studied (5-35 degrees C). This contribution of coenzyme binding energy to domain dynamics in CPR agrees with conclusions from previous temperature-jump studies [Gutierrez, A., Paine, M., Wolf, C. R., Scrutton, N. S., and Roberts, G. C. K. (2002) Biochemistry 41, 4626-4637]. A combination of calorimetry and stopped-flow spectrophotometry kinetics experiments showed that this linkage between coenzyme binding energetics and diffusional domain motion impinges directly on the molecular recognition of cytochrome c by CPR. Single-turnover reduction of cytochrome c by CPR (k(max) = 15 s(-)(1), K(d) = 37 microM) is critically coupled to coenzyme binding through ligand-induced motions that enable the FMN-binding domain to overcome a kinetically unproductive conformation. This is remarkable since the FMN-binding domain is not directly involved in coenzyme binding, the NADP(H) binding site being fully contained in the FAD-binding domain. Sequential rapid mixing measurements indicate that harnessing of coenzyme binding energy to the formation of a kinetically productive CPR-cytochrome c complex is a highly synchronized event. The inferred half-time for the decay of this productive conformation (tau(50)) is 330 +/- 70 ms only. Previously proposed structural and kinetic models are discussed in light of these findings.