Identity and function of an essential nitrogen ligand of the nitrogenase cofactor biosynthesis protein NifB.

NifB is a radical S-adenosyl-L-methionine (SAM) enzyme that is essential for nitrogenase cofactor assembly. Previously, a nitrogen ligand was shown to be involved in coupling a pair of [Fe4S4] clusters (designated K1 and K2) concomitant with carbide insertion into an [Fe8S9C] cofactor core (designated L) on NifB. However, the identity and function of this ligand remain elusive. Here, we use combined mutagenesis and pulse electron paramagnetic resonance analyses to establish histidine-43 of Methanosarcina acetivorans NifB (MaNifB) as the nitrogen ligand for K1. Biochemical and continuous wave electron paramagnetic resonance data demonstrate the inability of MaNifB to serve as a source for cofactor maturation upon substitution of histidine-43 with alanine; whereas x-ray absorption spectroscopy/extended x-ray fine structure experiments further suggest formation of an intermediate that lacks the cofactor core arrangement in this MaNifB variant. These results point to dual functions of histidine-43 in structurally assisting the proper coupling between K1 and K2 and concurrently facilitating carbide formation via deprotonation of the initial carbon radical

Structure of Precursor-Bound NifEN: A Nitrogenase FeMo Cofactor Maturase/Insertase

NifEN plays an essential role in the biosynthesis of the nitrogenase iron-molybdenum (FeMo) cofactor (M cluster). It is an α_2β_2 tetramer that is homologous to the catalytic molybdenum-iron (MoFe) protein (NifDK) component of nitrogenase. NifEN serves as a scaffold for the conversion of an iron-only precursor to a matured form of the M cluster before delivering the latter to its target location within NifDK. Here, we present the structure of the precursor-bound NifEN of Azotobacter vinelandii at 2.6 angstrom resolution. From a structural comparison of NifEN with des-M-cluster NifDK and holo NifDK, we propose similar pathways of cluster insertion for the homologous NifEN and NifDK proteins

Factors associated with physiotherapy provision in a population of elderly nursing home residents; a cross sectional study

BACKGROUND: Although physiotherapy (PT) plays an important role in improving activities of daily living (ADL functioning) and discharge rates, it is unclear how many nursing home residents receive treatment. Furthermore, there is a lack of insight into the determinants that influence the decision for treatment. In this study, we investigated how many nursing home residents receive PT. In addition, we analysed the factors that contribute to the variation in the provision of PT both between nursing homes and between residents. METHODS: A random sample of 600 elderly residents was taken from a random sample of 15 nursing homes. Residents had to be admitted for rehabilitation or for long-term care. Data were collected through interviews with the nursing home physician and the physiotherapist. Multilevel analysis was used to define the variation in the provision of PT and the factors that are associated with the question whether a resident receives PT or not. Furthermore the amount of PT provided was analysed and the factors that are associated with this. RESULTS: On average 69% of the residents received PT. The percentage of patients receiving treatment differed significantly across nursing homes, and especially the number of physiotherapists available, explained this difference between nursing homes. Residents admitted to a somatic ward for rehabilitation, and male residents in general, were most likely to receive PT. Residents who were treated by a physiotherapist received on average 55 minutes (sd 41) treatment a week. Residents admitted for rehabilitation received more PT a week, as were residents with a status after a total hip replacement. CONCLUSION: PT is most likely to be provided to residents on a somatic ward, recently admitted for rehabilitation to a nursing home, which has a relatively large number of physiotherapists. This suggests a potential under-use of PT for long-term residents with cognitive problems. It is recommended that physiotherapists reconsider which residents may benefit from treatment. This may require a shift in the focus of physiotherapists from 'recovery and discharge' to 'quality of life and well-being'

Caregivers' understanding of dementia predicts patients' comfort at death: a prospective observational study

Background: Patients with dementia frequently do not receive adequate palliative care which may relate to poor understanding of the natural course of dementia. We hypothesized that understanding that dementia is a progressive and terminal disease is fundamental to a focus on comfort in dementia, and examined how family and professional caregivers' understanding of the nature of the disease was associated with patients' comfort during the dying process. Methods: We enrolled 372 nursing home patients from 28 facilities in The Netherlands in a prospective observational study (2007 to 2010). We studied both the families and the physicians (73) of 161 patients. Understanding referred to families' comprehension of complications, prognosis, having been counseled on these, and perception of dementia as "a disease you can die from" (5-point agreement scale) at baseline. Physicians reported on this perception, prognosis and having counseled on this. Staff-assessed comfort with the End-of-Life in Dementia - Comfort Assessment in Dying (EOLD-CAD) scale. Associations between understanding and comfort were assessed with generalized estimating equations, structural equation modeling, and mediator analyses. Results: A family's perception of dementia as "a disease you can die from" predicted higher patient comfort during the dying process (adjusted coefficient -0.8, 95% confidence interval (CI): -1.5; -0.06 point increment disagreement). Family and physician combined perceptions (-0.9, CI: -1.5; -0.2; 9-point scale) were also predictive, including in less advanced dementia. Forty-three percent of the families perceived dementia as a disease you can die from (agreed completely, partly); 94% of physicians did. The association between combined perception and higher comfort was mediated by the families' reporting of a good relationship with the patient and physicians' perception that good care was provided in the last week. Conclusions: Awareness of the terminal nature of dementia may improve patient comfort at the end of life. Educating families on the nature of dementia may be an important part of advance care planning

Insertion of heterometals into the NifEN-associated iron–molybdenum cofactor precursor

The cofactors of Mo-, V-, Fe-dependent nitrogenases are believed to be highly homologous in structure despite the different types of heterometals (Mo, V, and Fe) they contain. Previously, a precursor form of the FeMo cofactor (FeMoco) was captured on NifEN, a scaffold protein for FeMoco biosynthesis. This all-Fe precursor closely resembles the Fe/S core structure of the FeMoco and, therefore, could reasonably serve as a precursor for all nitrogenase cofactors. Here, we report the heterologous incorporation of V and Fe into the NifEN-associated FeMoco precursor. EPR and activity analyses indicate that V and Fe can be inserted at much reduced efficiencies compared with Mo, and incorporation of both V and Fe is enhanced in the presence of homocitrate. Further, native polyacrylamide gel electrophoresis experiments suggest that NifEN undergoes a significant conformational rearrangement upon metal insertion, which allows the subsequent NifEN–MoFe protein interactions and the transfer of the cofactor between the two proteins. The combined outcome of these in vitro studies leads to the proposal of a selective mechanism that is utilized in vivo to maintain the specificity of heterometals in nitrogenase cofactors, which is likely accomplished through the redox regulation of metal mobilization by different Fe proteins (encoded by nifH, vnfH, and anfH, respectively), as well as the differential interactions between these Fe proteins and their respective scaffold proteins (NifEN and VnfEN) in the Mo-, V-, and Fe-dependent nitrogenase systems

Structural Analysis of a Nitrogenase Iron Protein from Methanosarcina acetivorans: Implications for CO2 Capture by a Surface-Exposed [Fe4S4] Cluster.

Nitrogenase iron (Fe) proteins reduce CO2 to CO and/or hydrocarbons under ambient conditions. Here, we report a 2.4-Å crystal structure of the Fe protein from Methanosarcina acetivorans (MaNifH), which is generated in the presence of a reductant, dithionite, and an alternative CO2 source, bicarbonate. Structural analysis of this methanogen Fe protein species suggests that CO2 is possibly captured in an unactivated, linear conformation near the [Fe4S4] cluster of MaNifH by a conserved arginine (Arg) pair in a concerted and, possibly, asymmetric manner. Density functional theory calculations and mutational analyses provide further support for the capture of CO2 on MaNifH while suggesting a possible role of Arg in the initial coordination of CO2 via hydrogen bonding and electrostatic interactions. These results provide a useful framework for further mechanistic investigations of CO2 activation by a surface-exposed [Fe4S4] cluster, which may facilitate future development of FeS catalysts for ambient conversion of CO2 into valuable chemical commodities.IMPORTANCE This work reports the crystal structure of a previously uncharacterized Fe protein from a methanogenic organism, which provides important insights into the structural properties of the less-characterized, yet highly interesting archaeal nitrogenase enzymes. Moreover, the structure-derived implications for CO2 capture by a surface-exposed [Fe4S4] cluster point to the possibility of developing novel strategies for CO2 sequestration while providing the initial insights into the unique mechanism of FeS-based CO2 activation

The FeMoco-deficient MoFe Protein Produced by a nifH Deletion Strain of Azotobacter vinelandii Shows Unusual P-cluster Features

The His-tag MoFe protein expressed by the nifH deletion strain Azotobacter vinelandii DJ1165 (Delta nifH MoFe protein) was purified in large quantity. The alpha 2beta 2 tetrameric Delta nifH MoFe protein is FeMoco-deficient based on metal analysis and the absence of the S = 3/2 EPR signal, which arises from the FeMo cofactor center in wild-type MoFe protein. The Delta nifH MoFe protein contains 18.6 mol Fe/mol and, upon reduction with dithionite, exhibits an unusually strong S = 1/2 EPR signal in the g approx 2 region. The indigo disulfonate-oxidized Delta nifH MoFe protein does not show features of the P2+ state of the P-cluster of the Delta nifB MoFe protein. The oxidized Delta nifH MoFe protein is able to form a specific complex with the Fe protein containing the [4Fe-4S]1+ cluster and facilitates the hydrolysis of MgATP within this complex. However, it is not able to accept electrons from the [4Fe-4S]1+ cluster of the Fe protein. Furthermore, the dithionite-reduced Delta nifH MoFe can be further reduced by Ti(III) citrate, which is quite unexpected. These unusual catalytic and spectroscopic properties might indicate the presence of a P-cluster precursor or a P-cluster trapped in an unusual conformation or oxidation state

Cofactor specificity motifs and the induced fit mechanism in class I ketol-acid reductoisomerases

Although most sequenced members of the industrially important ketol-acid reductoisomerase (KARI) family are class I enzymes, structural studies to date have focused primarily on the class II KARIs, which arose through domain duplication. In the present study, we present five new crystal structures of class I KARIs. These include the first structure of a KARI with a six-residue β2αB (cofactor specificity determining) loop and an NADPH phosphate-binding geometry distinct from that of the seven- and 12-residue loops. We also present the first structures of naturally occurring KARIs that utilize NADH as cofactor. These results show insertions in the specificity loops that confounded previous attempts to classify them according to loop length. Lastly, we explore the conformational changes that occur in class I KARIs upon binding of cofactor and metal ions. The class I KARI structures indicate that the active sites close upon binding NAD(P)H, similar to what is observed in the class II KARIs of rice and spinach and different from the opening of the active site observed in the class II KARI of Escherichia coli. This conformational change involves a decrease in the bending of the helix that runs between the domains and a rearrangement of the nicotinamide-binding site

De relatie van psychiatrische functiestoornissen met cognitie, activiteiten dagelijks leven (ADL) en biografische gegevens

Probleemstelling: Psychiatrische functiestoornissen (PFS) hebben een negatief effect op de kwaliteit van leven van psychogeriatrische patiënten, op hun levensduur en op het mantelzorgsysteem. Ook zijn PFS voorspellers voor zowel permanente opname als voor de kans op ontslag. De prevalentie van PFS bij psychogeriatrische patiënten in een verpleeghuis ligt tussen 70 en 80%. In een eerder onderzoek werden relatief zwakke relaties gevonden tussen PFS en cognitieve functiestoornissen, zelfzorg-problemen en biografische gegevens. Dit onderzoek wordt hier gerepliceerd. De prevalentie van PFS is onderzocht bij psychogeriatrische patiënten die zich aanmeldden voor onderzoek op een polikliniek voor cognitieve functiestoornissen. Onderzoek en methode: In de Indexconditie participeerden 70 patiënten (leeftijd ≥ 65) met klachten over cognitief functioneren die verwezen werden naar een polikliniek voor cognitieve functiestoornissen in de regio Nieuwe Waterweg Noord en van wie vermoed werd dat zij leden aan aanmerkelijke psychiatrische functiestoornissen. Van 35 patiënten waren volledige data op NPI, MMSE en Barthel Index (BI) beschikbaar. In de Referentieconditie participeerden 487 patiënten (leeftijd ≥ 65) uit de regio Nieuwe Waterweg Noord, verwezen voor trans- of intramurale verpleeghuiszorg en van wie vermoed werd dat zij leden aan cognitieve functiestoornissen (MMSE ≥ 29). Van 385 patiënten waren alle data beschikbaar op NPI, MMSE en BI. Resultaten: Van de patiënten had 92% minimaal 1 symptoom score of meer op de NPI; 82% had twee of meer symptomen. De NPI-symptomen Depressie, Apathie, Angst en Prikkelbaarheid toonden een hoge prevalentie in beide steekproeven. Logistische regressieanalyse liet zien dat MMSE, BI en biografische gegevens een geringe voorspellende waarde hadden voor zowel de totale NPI-score als voor afzonderlijke NPI-symptomen (R2 =0,11; max). Een niet-metrische principale componentenanalyse en een confirmatieve factoranalyse toonden aan dat de relaties tussen NPI, MMSE en BI in de twee steekproeven niet significant verschilden. MMSE en BI correleerden in de twee steekproeven met de dimensie ‘Cognitie’, en NPI met de dimensie ‘Psychiatrische functiestoornis’. Conclusie: In de Indexconditie (N=35) werden de bevindingen uit de Referentieconditie (N=385) bevestigd. Dit betrof de hoogte van de frequentie van PFS (NPI), de brede spreiding over de NPI-symptomen; de minimale prognostische rol van MMSE, BI en biografische gegevens voor zowel de totale NPI-score als de individuele NPI-symptomen. De PFS vormden ook in de Indexconditie een dimensie op zich. Met name bij verwijzing en indicatiestelling voor behandeling en zorg is het van belang een onderscheid te maken tussen de psychiatrische en de cognitieve dimensie