Blood-sucking Insect Rhodnius Prolixus Research Articles

Electrostatic Tuning of the Ligand Binding Mechanism by Glu27 in Nitrophorin 7

Nitrophorins (NP) 1–7 are NO-carrying heme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus. The isoform NP7 displays peculiar properties, such as an abnormally high isoelectric point, the ability to bind negatively charged membranes, and a strong pH sensitivity of NO affinity. A unique trait of NP7 is the presence of Glu in position 27, which is occupied by Val in other NPs. Glu27 appears to be important for tuning the heme properties, but its influence on the pH-dependent NO release mechanism, which is assisted by a conformational change in the AB loop, remains unexplored. Here, in order to gain insight into the functional role of Glu27, we examine the effect of Glu27 → Val and Glu27 → Gln mutations on the ligand binding kinetics using CO as a model. The results reveal that annihilation of the negative charge of Glu27 upon mutation reduces the pH sensitivity of the ligand binding rate, a process that in turn depends on the ionization of Asp32. We propose that Glu27 exerts a through-space electrostatic action on Asp32, which shifts the pKa of the latter amino acid towards more acidic values thus reducing the pH sensitivity of the transition between open and closed states.

Adipokinetic hormone receptor gene identification and its role in triacylglycerol metabolism in the blood-sucking insect Rhodnius prolixus

Adipokinetic hormone (AKH) has been associated with the control of energy metabolism in a large number of arthropod species due to its role on the stimulation of lipid, carbohydrate and amino acid mobilization/release. In the insect Rhodnius prolixus, a vector of Chagas' disease, triacylglycerol (TAG) stores must be mobilized to sustain the metabolic requirements during moments of exercise or starvation. Besides the recent identification of the R. prolixus AKH peptide, other components required for the AKH signaling cascade and its mode of action remain uncharacterized in this insect. In the present study, we identified and investigated the expression profile of the gene encoding the AKH receptor of R. prolixus (RhoprAkhr). This gene is highly conserved in comparison to other sequences already described and its transcript is abundant in the fat body and the flight muscle of the kissing bug. Moreover, RhoprAkhr expression is induced in the fat body at moments of increased TAG mobilization; the knockdown of this gene resulted in TAG accumulation both in fat body and flight muscle after starvation. The inhibition of Rhopr-AKHR transcription as well as the treatment of insects with the peptide Rhopr-AKH in its synthetic form altered the transcript levels of two genes involved in lipid metabolism, the acyl-CoA-binding protein-1 (RhoprAcbp1) and the mitochondrial glycerol-3-phosphate acyltransferase-1 (RhoprGpat1). These results indicate that the AKH receptor is regulated at transcriptional level and is required for TAG mobilization under starvation. In addition to the classical view of AKH as a direct regulator of enzymatic activity, we propose here that AKH signaling may account for the regulation of nutrient metabolism by affecting the expression profile of target genes.

Comparing de novo and reference-based transcriptome assembly strategies by applying them to the blood-sucking bug Rhodnius prolixus

High Throughput Sequencing capabilities have made the process of assembling a transcriptome easier, whether or not there is a reference genome. But the quality of a transcriptome assembly must be good enough to capture the most comprehensive catalog of transcripts and their variations, and to carry out further experiments on transcriptomics. There is currently no consensus on which of the many sequencing technologies and assembly tools are the most effective.Many non-model organisms lack a reference genome to guide the transcriptome assembly. One question, therefore, is whether or not a reference-based genome assembly gives better results than de novo assembly. The blood-sucking insect Rhodnius prolixus—a vector for Chagas disease—has a reference genome. It is therefore a good model on which to compare reference-based and de novo transcriptome assemblies.In this study, we compared de novo and reference-based genome assembly strategies using three datasets (454, Illumina, 454 combined with Illumina) and various assembly software. We developed criteria to compare the resulting assemblies: the size distribution and number of transcripts, the proportion of potentially chimeric transcripts, how complete the assembly was (completeness evaluated both through CEGMA software and R. prolixus proteome fraction retrieved). Moreover, we looked for the presence of two chemosensory gene families (Odorant-Binding Proteins and Chemosensory Proteins) to validate the assembly quality. The reference-based assemblies after genome annotation were clearly better than those generated using de novo strategies alone. Reference-based strategies revealed new transcripts, including new isoforms unpredicted by automatic genome annotation. However, a combination of both de novo and reference-based strategies gave the best result, and allowed us to assemble fragmented transcripts.

Oviposition in the blood-sucking insect Rhodnius prolixus is modulated by host odors.

BackgroundTriatomine bugs are blood-sucking insects, vectors of Chagas disease. Despite their importance, their oviposition behavior has received relatively little attention. Some triatomines including Rhodnius prolixus stick their eggs to a substrate. It is known that mechanical cues stimulate oviposition in this species. However, it is not clear if chemical signals play a role in this behavior. We studied the role of host cues, including host odor, in the oviposition behavior of the triatomine R. prolixus.MethodsTests were carried out in an experimental arena and stimuli consisted of a mouse or hen feathers. The number of eggs laid and the position of those eggs with respect to the stimulus source were recorded. Data were analyzed using the Mann-Whitney and Kruskal-Wallis tests.ResultsBoth a mouse and hen feathers stimulated oviposition. In addition, hen feathers evoked a particular spatial distribution of eggs that was not observed in the case of mouse.ConclusionsWe propose that volatile chemical cues from the host play a role in the oviposition behavior of triatomines that stick their eggs. Thus, host odor would stimulate and spatially guide oviposition.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0867-5) contains supplementary material, which is available to authorized users.

Rhodnius respiration depends on multiple factors

Sucking great lungfuls of air into the body is the main response of most mammals to physical exertion. As their metabolic rates rise, they inhale more deeply to maintain power. However, for insects, breathing is more complex. Equipped with a network of fine ventilation tubes that permeate every tissue in the body, insects have to tightly regulate the passage of oxygen into the body as their metabolic demands increase, by altering their breathing patterns. Timothy Bradley from the University of California, Irvine, USA, notes that respiratory patterns were thought to be principally determined by metabolic rate. But bugs don't just ramp up their metabolism in response to exercise; their metabolic rates increase as temperatures rise and during reproduction and digestion, so it wasn't clear whether other activities might result in different respiratory patterns. ‘There was some evidence that the respiratory control mechanism might be temperature sensitive’, says graduate student Erica Heinrich. She and Bradley began investigating the effects of feeding and temperature on the blood-sucking insect Rhodnius prolixus (p. 2752).‘Rhodnius prolixus provides certain valuable features as a model insect for studying respiratory control’, says Heinrich, who explains that the insect's metabolic rate rockets after dining on blood. This allowed her to measure the metabolic rates and respiratory patterns of the insects as they exhaled CO2 after a satisfying rabbit blood meal, in addition to monitoring the effects of temperature on the unfed insects as she varied the temperature between 18 and 38°C.Sure enough, as the temperature rose, the insects' metabolic rate increased 3.5 times above their resting metabolic rate and, as their metabolic demands increased, they initially opened and closed their spiracle valves (breathed discontinuously) until the metabolic rate became high enough and the spiracles remained open continuously.Next, the duo measured the metabolic rates of the well-fed insects and they were amazed to see the bugs' metabolic rates hurtle to almost 14 times their resting metabolic rates. However, when they analysed the bugs' CO2 exhalation pattern, they found, surprisingly, that instead of holding the spiracles open and exhaling CO2 continuously, they continued closing the spiracles intermittently, even though the insects' metabolic rate was four times higher than that at the hottest temperature.The duo also monitored the amount of CO2 released by the insects during each exhalation burst and found that they exhaled less CO2 per breath as the temperature rose. This was in contrast to the digesting insects, which exhaled more CO2 each time they opened their spiracles as their metabolic rates rose.Having shown that the insects' respiratory pattern was different under the two situations, Bradley says, ‘It is overly simplistic to attribute respiratory pattern to metabolic rate alone’, and the duo suspects that the insects' respiratory system is temperature sensitive. Explaining that if the respiration system was not sensitive to temperature, insects should always exhale the same amount of CO2 each time that open their spiracles, Heinrich says, ‘However, we found that as the temperature increases, the volume of CO2 released in a burst decreases’. She and Bradley suspect that this temperature sensitivity may result from changes in the pH of water in the insect's body. ‘Increased temperature decreases the pH of water’, explains Heinrich. She adds, ‘If the CO2 threshold that triggers spiracle opening is sensed via pH, then exposure to high temperatures will trigger premature spiracle opening… and less CO2 would need to accumulate via metabolism to reach the pH threshold. This will result in reduced volumes of CO2 release during spiracle opening’.

Identification of uncoupling protein 4 from the blood-sucking insect Rhodnius prolixus and its possible role on protection against oxidative stress

Uncoupling proteins (UCPs) play a critical role in the control of the mitochondrial membrane potential (ΔΨm) due to their ability to dissipate the proton gradient, which results in the uncoupling of mitochondrial respiration from ATP production. Most reactive oxygen species generation in mitochondria occurs in complex III, due to an increase of semiquinone (Q−) half-life. When active, UCPs can account as a potential antioxidant system by decreasing ΔΨm and increasing mitochondrial respiration, thus reducing Q− life time. The hematophagous insect Rhodnius prolixus, a vector of Chagas disease, is exposed to a huge increase in oxidative stress after a blood meal because of the hydrolysis of hemoglobin and the release of the cytotoxic heme molecule. Although some protective mechanisms were already described for this insect and other hematophagous arthropods, the putative role of UCP proteins as antioxidants in this context has not been explored. In this report, two genes encoding UCP proteins (RpUcp4 and RpUcp5) were identified in the R. prolixus genome. RpUcp4 is the predominant transcript in most analyzed organs, and both mRNA and protein expression are upregulated (13- and 3-fold increase, respectively) in enterocytes the first day after the blood feeding. The increase in UCP4 expression is coincident with the decrease in hydrogen peroxide (H2O2) generation by midgut cells. Furthermore, in mitochondria isolated from enterocytes, the modulation of UCP activity by palmitic acid and GDP resulted in altered ΔΨm, as well as modulation of H2O2 generation rates. These results indicate that R. prolixus UCP4 may function in an antioxidation mechanism to protect the midgut cells against oxidative damage caused by blood digestion.

Nitric oxide heme interactions in nitrophorin 7 investigated by nuclear inelastic scattering

Nitrophorins (NPs) occur in the blood-sucking insect Rhodnius prolixus. These proteins use ferric heme to store nitric oxide (NO) in the salivary glands of the insects and transport it to the victim’s tissues, resulting in vasodilation and reduced blood coagulation. In this work we present a nuclear inelastic scattering (NIS) study in order to characterize the iron-NO interaction in the isoform nitrophorin 7 (NP7). The NIS data obtained for NP7 complexed with NO show a strong band at ∼589 cm−1 which is due to modes with significant Fe-NO stretching and bending character. Another conspicuous feature is a significant peak at ∼280 cm−1 in the region where the heme modes occur. Based on a hybrid calculation method, which uses density functional theory and molecular mechanics, the band at ∼280 cm−1 is assigned to heme modes with substantial doming character.

NMR studies of the dynamics of nitrophorin 2 bound to nitric oxide.

The Rhodnius nitrophorins are β-barrel proteins of the lipocalin fold with a heme protruding from the open end of the barrel. They are found in the saliva of the blood-sucking insect Rhodnius prolixus, which synthesizes and stores nitric oxide (NO) in the salivary glands, where NO is bound to iron. NO is released by dilution and an increase in pH when the insect spits its saliva into the tissues of a victim, to aid in obtaining a blood meal. In the adult insect, there are four nitrophorins, NP1-NP4. At pH 7.3, NP4 releases NO 17 times faster than NP2 does, as measured by stopped-flow kinetics. A number of crystal structures of the least abundant protein, NP4, are available. These structures have been used to propose that two loops between adjacent β-strands at the front opening of the protein, the A-B and G-H loops, determine the rate of NO release. To learn how the protein loops contribute to the release of NO for each of the nitrophorins, the dynamics of these proteins are being studied in our laboratory. In this work, the NP2-NO complex has been investigated by nuclear magnetic resonance relaxation measurements to probe the picosecond-to-nanosecond and microsecond-to-millisecond time scale motions at three pH values, 5.0, 6.5, and 7.3. It is found that at pH 5.0 and 6.5, the NP2-NO complex is rigid and only a few residues in the loop regions show dynamics, while at pH 7.3, somewhat more dynamics, particularly of the A-B loop, are observed. Comparison to other lipocalins shows that all are relatively rigid, and that the dynamics of lipocalins in general are much more subtle than those of mainly α-helical proteins.

Expression, Purification, and Solid-State NMR Characterization of the Membrane Binding Heme Protein Nitrophorin 7 in Two Electronic Spin States

The nitrophorins (NPs) comprise a group of NO transporting ferriheme b proteins found in the saliva of the blood sucking insect Rhodnius prolixus . In contrast to other nitrophorins (NP1-4), the recently identified membrane binding isoform NP7 tends to form oligomers and precipitates at higher concentrations in solution. Hence, solid-state NMR (ssNMR) was employed as an alternative method to gain structural insights on the precipitated protein. We report the expression and purification of (13)C,(15)N isotopically labeled protein together with the first ssNMR characterization of NP7. Because the size of NP7 (21 kDa) still provides a challenge for ssNMR, the samples were reverse labeled with Lys and Val to reduce the number of crosspeaks in two-dimensional spectra. The two electronic spin states with S = 1/2 and S = 0 at the ferriheme iron were generated by the complexation with imidazole and NO, respectively. ssNMR spectra of both forms are well resolved, which allows for sequential resonance assignments of 22 residues. Importantly, the ssNMR spectra demonstrate that aggregation does not affect the protein fold. Comparison of the spectra of the two electronic spin states allows the determination of paramagnetically shifted cross peaks due to pseudocontact shifts, which assists the assignment of residues close to the heme center.

Characterization of burdock mottle virus, a novel member of the genus Benyvirus, and the identification of benyvirus-related sequences in the plant and insect genomes

The complete nucleotide sequence of the burdock mottle virus (BdMoV) isolated from an edible burdock plant (Arctium lappa) in Japan has been determined. BdMoV has a bipartite genome, whose organization is similar to RNA1 and RNA2 of benyviruses, beet necrotic yellow vein virus (BNYVV), beet soil-borne mosaic virus (BSBMV), and rice stripe necrosis virus (RSNV). BdMoV RNA1 (7038 nt) contains a single open reading frame (ORF) encoding a 249-kDa polypeptide that consists of methyl-transferase, helicase, papain-like protease, AlkB-like, and RNA-dependent RNA polymerase domains. The AlkB-like domain sequence is not present in the proteins encoded by other known benyviruses, but is found in replication-associated proteins of viruses mainly belonging to the families Alfaflexiviridae and Betaflexiviridae. BdMoV RNA2 (4315 nt) contains six ORFs that are similar to those of benyviruses: these are coat protein (CP), CP readthrough, triple gene block movement and cysteine-rich proteins. Phylogenetic analyses showed that BdMoV is more closely related to BNYVV and BSBMV than to RSNV. Database searches showed that benyvirus replicase-related sequences are present in the chromosomes of a chickpea plant (Cicer arietinum) and a blood-sucking insect (Rhodnius prolixus). Some other benyvirus-related sequences are found in the transcriptome shotgun libraries of a few species of plants and a bark beetle. Our results show that BdMoV is a distinct species of the genus Benyvirus and that ancestral and extant benyviruses may have infected or currently infect a wide range of hosts, including plants and insects.

Kinetics and computational studies of ligand migration in nitrophorin 7 and its Δ1–3 mutant

Nitrophorins (NPs) are nitric oxide (NO)-carrying heme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus. Though NP7 exhibits a large sequence resemblance with other NPs, two major differential features are the ability to interact with negatively charged cell surfaces and the presence of a specific N-terminus composed of three extra residues (Leu1-Pro2-Gly3). The aim of this study is to examine the influence of the N-terminus on the ligand binding, and the topological features of inner cavities in closed and open states of NP7, which can be associated to the protein structure at low and high pH, respectively. Laser flash photolysis measurements of the CO rebinding kinetics to NP7 and its variant NP7(Δ1–3), which lacks the three extra residues at the N-terminus, exhibit a similar pattern and support the existence of a common kinetic mechanism for ligand migration and binding. This is supported by the existence of a common topology of inner cavities, which consists of two docking sites in the heme pocket and a secondary site at the back of the protein. The ligand exchange between these cavities is facilitated by an additional site, which can be transiently occupied by the ligand in NP7, although it is absent in NP4. These features provide a basis to explain the enhanced internal gas hosting capacity found experimentally in NP7 and the absence of ligand rebinding from secondary sites in NP4. The current data allow us to speculate that the processes of docking to cell surfaces and NO release may be interconnected in NP7, thereby efficiently releasing NO into a target cell. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

P53: Nitrite dismutase – A “nitrite-only” source for NO

Background Nitrophorins (NPs) are NO transporting proteins from the saliva of the blood-sucking insect Rhodnius prolixus . The delivery of NO is accomplished by storage on the protein’s ferriheme cofactor and the gas molecule is released when the protein is injected into a victim’s tissue. However, we demonstrated that nitrophorins can also produce NO from nitrite as the only substrate, i.e., in this reaction nitrite serves as both electron donor and electron acceptor. The enzymatic activity was termed nitrite dismutase activity and is now classified with the EC number 1.7.6.1. This study provides insight into the structural features required for the catalytic activity. Methods Proteins were recombinantly expressed in Escherichia coli cells. They were characterized by X-ray crystallography, EPR spectroscopy, UV/vis spectroscopy, resonance Raman spectroscopy, and NMR spectroscopy. The enzymatic reaction was studied by absorbance detected stopped-flow kinetics. Results The binding mode of nitrite to the NP Fe (III) is clearly different compared to that of the globins, i.e., N-bound. Contrary to the globins, the insertion of an H-donating residue (NP4(L130R)) into the protein pocket does not affect the nitrite binding mode; however, it cancels the enzymatic activity. On the other hand, mutation of the proximal Asp70 residue, which is H-bonded via a water to the iron coordinating His59, also affects the enzymatic activity, indicating that the charge distribution on the heme cofactor has a major influence on the iron reactivity. Conclusion The nitrophorins are good model systems for the characterization of the nitrite dismutase catalysis. The absence of H-donating residues and the charge density on the proximal His were identified as important structural features that are required for the nitrite dismutase reaction. Among the many heme proteins expressed in the human organism some might bear a similar catalytic activity. We suggest the group of heme peroxidases as potential candidates for enzymatic “nitrite-only” NO production. Disclosure Supported by the Deutsche Forschungsgemeinschaft (DFG), Grants KN 951-1/1 and 2.

Complexes of ferriheme nitrophorin 4 with low-molecular weight thiol(ate)s occurring in blood plasma

Nitrophorins are proteins occurring in the saliva of the blood-sucking insect Rhodnius prolixus to carry NO as a vasodilator and blood-coagulation inhibitor into the victim's tissue. It was suggested that the rate of NO release can be enhanced by the blood-plasma component l-cysteine [J.M.C.Ribeiro, Insect Biochem. Mol. Biol. 26 (1996) 899–905]. However, the mechanism of the reaction is not clear. In the attempt to exploit the reaction in detail, complexes of nitrophorin 4 (NP4) with the thiols 2-mercaptoethanol, l-cysteine, and l-homocysteine and with HS− were formed and characterized under anaerobic conditions using absorption spectroscopy, X-ray crystallography, and EPR spectroscopy. In contrast to met-myoglobin, which is reduced by l-cysteine, all four compounds form low-spin FeIII complexes with NP4. The weak equilibration constants (167–5200M−1) neither support significant complexation nor the simple displacement of NO in vivo. Both amino acid based thiols form additional H-bonds with side chains of the heme pocket entry. Glutathione and l-methionine did not form a complex, indicating the specificity of the complexes with l-cysteine and l-homocysteine. Continuous wave EPR spectroscopy reveals the simultaneous existence of three low-spin systems in each case that are attributed to various protonation and/or conformational stages in the heme pocket. Electron nuclear double resonance (ENDOR) spectroscopy demonstrates that the thiol sulfurs are, at least in part, protonated. Overall, the results not only demonstrate the good accessibility of the NP4 heme center by biologically relevant thiols, but also represent the first structural characterization of a ferriheme protein in complex with l-cysteine l-homocysteine.

Erratum to: Assignment of the 1H NMR resonances of protein residues in close proximity to the heme of the nitrophorins: similarities and differences among the four proteins from the saliva of the adult blood-sucking insect Rhodnius prolixus

Erratum to: Assignment of the 1H NMR resonances of protein residues in close proximity to the heme of the nitrophorins: similarities and differences among the four proteins from the saliva of the adult blood-sucking insect Rhodnius prolixus

Assignment of the 1H NMR resonances of protein residues in close proximity to the heme of the nitrophorins: similarities and differences among the four proteins from the saliva of the adult blood-sucking insect Rhodnius prolixus

The nuclear Overhauser effects (NOEs) observed between heme substituent protons and a small number of nearby protein side chain protons in the water-elimination Fourier transform NOE spectroscopy (WEFT-NOESY) spectra of high- and low-spin wild-type nitrophorin (NP) 2 and its ligand complexes have been analyzed and compared with those observed for the same complexes of wild-type NP3. These assignments were made on naturally abundant isotope samples, with the most useful protein side chains being those of Ile120, Leu122, and Leu132 for NP2 and NP3, and Thr121, Leu123, and Leu133 for NP1 and NP4. It is found that the NOEs observed are identical, with extremely similar protein side chain proton chemical shifts. This is strong evidence that the structure of NP3, for which no X-ray crystal structures are available, is essentially identical to that of NP2, at least near the heme binding pocket. Similarly, the NOEs observed between heme substituents and protein side chains for NP1 and NP4 also indicate that the structures of the protein having both A and B heme orientations are very similar to each other, as well as to the proteins with major B heme orientation of NP2 and NP3. These A and B connectivities can be seen, even though the two heme orientations have similar populations in NP1 and NP4, which complicates the analysis of the NOESY spectra. The histamine complex of wild-type NP2 shows significant shifts of the Leu132 side chain protons relative to all other ligand complexes of NP1-NP4 because of the perturbation of the structure near Leu132 caused by the histamine's side chain ammonium hydrogen bond to the Asp29 side chain carboxylate.

Nuclear Inelastic Scattering and Mössbauer Spectroscopy as Local Probes for Ligand Binding Modes and Electronic Properties in Proteins: Vibrational Behavior of a Ferriheme Center inside a β-Barrel Protein

In this work, we present a study of the influence of the protein matrix on its ability to tune the binding of small ligands such as NO, cyanide (CN(-)), and histamine to the ferric heme iron center in the NO-storage and -transport protein Nitrophorin 2 (NP2) from the salivary glands of the blood-sucking insect Rhodnius prolixus. Conventional Mössbauer spectroscopy shows a diamagnetic ground state of the NP2-NO complex and Type I and II electronic ground states of the NP2-CN(-) and NP2-histamine complex, respectively. The change in the vibrational signature of the protein upon ligand binding has been monitored by Nuclear Inelastic Scattering (NIS), also called Nuclear Resonant Vibrational Spectroscopy (NRVS). The NIS data thus obtained have also been calculated by quantum mechanical (QM) density functional theory (DFT) coupled with molecular mechanics (MM) methods. The calculations presented here show that the heme ruffling in NP2 is a consequence of the interaction with the protein matrix. Structure optimizations of the heme and its ligands with DFT retain the characteristic saddling and ruffling only if the protein matrix is taken into account. Furthermore, simulations of the NIS data by QM/MM calculations suggest that the pH dependence of the binding of NO, but not of CN(-) and histamine, might be a consequence of the protonation state of the heme carboxyls.

Recent experiments towards a model for fluid secretion in Rhodnius Upper Malpighian Tubules (UMT)

Three different methods have been used to improve a model for fluid secretion in Upper Malpighian Tubules (UMT) of the blood sucking insect Rhodnius prolixus. (I) In the first, UMT double perfusions in 5th instar Rhodnius were used to measure their fluid secretion rate. They were stimulated to secrete with 5-HT. Double perfusions allowed access separately to the basolateral and the apical cell membranes with pharmacological agents known to block different ion transport functions, namely ATPases, cotransporters and/or countertransporters and ion and water channels: ouabain, bafilomycin A1, furosemide, bumetanide, SITS, acetazolamide, amiloride, DPC, BaCl2, pCMBS and DTT. The basic assumption is that changes in water movement reflect changes in ion transport mechanisms. (II) Intracellular Na+ concentrations were measured with a fluorometric method in dissected R. prolixus UMT, under several experimental conditions. (III) ATPase activities were measured in R. prolixus UMT. A tentative model for the function of the UMT cell is presented. We find that (a) at the basolateral cell membrane, fundamental is a Na+–K+–2Cl− cotransporter; of intermediate importance are the Na+–K+-ATPase and a ouabain-insensitive Na+-ATPase, ion channels and Rp-MIP water channels. (b) At the apical cell membrane, most important are a V-H+-ATPase; and a K+ and/or Na+–H+ exchanger.

Nuclear inelastic scattering of heme proteins: from iron ligand vibrations to low energy protein modes

The binding of the signal molecule nitric oxide (NO) to the NO transporter protein Nitrophorin 2 (NP2) from the bloodsucking insect Rhodnius prolixus has been characterized by Mossbauer spectroscopy as well as nuclear forward scattering (NFS) and nuclear inelastic scattering (NIS). A striking feature of the vibrational spectrum obtained from NP2-NO is a vibration at 594 cm − 1. This mode is assigned to a Fe-NO stretching mode via simulation of the NIS data by density functional theory (DFT) coupled with molecular mechanics (MM) methods. At frequencies below 100 cm − 1 collective motions like heme doming occur which could explain spectroscopic features observed by NIS at these low energies.

NMR studies of nitrophorin distal pocket side chain effects on the heme orientation and seating of NP2 as compared to NP1

The nitrophorins (NP) of the adult blood-sucking insect Rhodnius prolixus fall into two pairs based on sequence identity (NP1,4 (90%) and NP2,3 (79%)), which differ significantly in the size of side chains of residues which contact the heme. These residues include those in the distal pocket of NP2 (I120) and NP1 (T121) and the “belt” that surrounds the heme of NP2 (S40, F42), and NP1(A42, L44). To determine the importance of these residues and others conserved or very similar for the two pairs, including L122(123), L132(133), appropriate mutants of NP2 and NP1 have been prepared and studied by 1H NMR spectroscopy. Wild-type NP2 has heme orientation ratio ( A: B) of 1:8 at equilibrium, while wild-type NP1 has A: B ~ 1:1 at equilibrium. Another difference between NP2 and NP1 is in the heme seating with regard to His57(59). It is found that among the distal pocket residues investigated, the residue most responsible for heme orientation and seating is I120(T121). F42(L44) and L106(F107) may also be important, but must be investigated in greater detail.

Nitrophorins: Nitrite disproportionation reaction and other novel functionalities of insect heme‐based nitric oxide transport proteins

Nitrophorins (NPs) comprise a unique class of heme proteins used by the blood-sucking insect Rhodnius prolixus to deliver the signaling gas molecule NO into the blood vessel of a host during feeding. Upon NO release, histamine can be scavenged by coordination to the heme iron. Although the protein is of similar size as the mammalian globin monomers and shares the same cofactor and proximal histidine coordination, nitrophorin structure, in contrast, is almost entirely composed of a β-barrel. Comparison of the NO and histamine association constants with the concentrations of both compounds invivo raises concerns about the very simple ligand release model in case of at least some of the NPs. Therefore, novel functionalities of the NPs were sought. As a result, catalysis of the nitrite disproportionation reaction was found, which leads to the formation of NO with nitrite as the sole substrate. This is the first example of a ferriheme protein that can perform this reaction. Furthermore, although NPs stabilize the ferriheme state, a peroxidase reactivity of the cofactor involving the higher oxidation state iron (Compound I/II) was studied with the potential to catalyze the oxidation of histamine and norepinephrine. In contrast to many other heme proteins including the globins, the ferroheme state was found to be extremely sensitive to O(2) , which is a consequence of the much lower reduction potential of the NPs, so that the 1-electron reduction of O(2) to O (•-)(2) becomes a thermodynamically favored process. Altogether, the detailed study of the NPs gives insight into the structure-function relationships required for the targeted delivery of diatomic gas molecules in biology. Moreover, the comparison of the structure-function relationships of the NPs (NO transporters) with those of the globins (O(2) transporters) will help to elucidate the architectural requirement for the respective tasks.