How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase

Salmonella enterica is an opportunistic pathogen that produces a [NiFe]-hydrogenase under aerobic conditions. In the present study, genetic engineering approaches were used to facilitate isolation of this enzyme, termed Hyd-5. The crystal structure was determined to a resolution of 3.2 Å and the hydro-genase was observed to comprise associated large and small subunits. The structure indicated that His(229) from the large subunit was close to the proximal [4Fe–3S] cluster in the small subunit. In addition, His(229) was observed to lie close to a buried glutamic acid (Glu(73)), which is conserved in oxygen-tolerant hydrogenases. His(229) and Glu(73) of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygen-tolerance mechanism. Substitution of His(229) or Glu(73) with alanine led to a loss in the ability of Hyd-5 to oxidize hydrogen in air. Furthermore, the H229A variant was found to have lost the overpotential requirement for activity that is always observed with oxygen-tolerant [NiFe]-hydrogenases. It is possible that His(229) has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu(73)could play a supporting role in fine-tuning the chemistry of His(229) to enable this function

Interview with Melissa & Rod Volbeda, Willamette Valley Cheese, 2007 (audio)

Interview of Melissa and Rod Volbeda by Magda Gaytan on August 6th, 2007. Willamette Valley Cheese Company tour photos available for download

How the oxygen tolerance of a [NiFe]-hydrogenase depends on quaternary structure

‘Oxygen-tolerant’ [NiFe]-hydrogenases can catalyze H(2) oxidation under aerobic conditions, avoiding oxygenation and destruction of the active site. In one mechanism accounting for this special property, membrane-bound [NiFe]-hydrogenases accommodate a pool of electrons that allows an O(2) molecule attacking the active site to be converted rapidly to harmless water. An important advantage may stem from having a dimeric or higher-order quaternary structure in which the electron-transfer relay chain of one partner is electronically coupled to that in the other. Hydrogenase-1 from E. coli has a dimeric structure in which the distal [4Fe-4S] clusters in each monomer are located approximately 12 Å apart, a distance conducive to fast electron tunneling. Such an arrangement can ensure that electrons from H(2) oxidation released at the active site of one partner are immediately transferred to its counterpart when an O(2) molecule attacks. This paper addresses the role of long-range, inter-domain electron transfer in the mechanism of O(2)-tolerance by comparing the properties of monomeric and dimeric forms of Hydrogenase-1. The results reveal a further interesting advantage that quaternary structure affords to proteins

Renal function in critically ill patients: assessment, replacement and histopathology. Towards personalized care

Patients admitted to the Intensive Care Unit (ICU) frequently develop acute kidney injury (AKI). Changes in renal function are evaluated by blood levels of creatinine. Creatinine is a breakdown product of muscle cells which is eliminated by the kidneys via the urine. Renal function loss leads to accumulation of waste products and fluids, which may cause critical metabolic derangements. Patients with AKI may need renal replacement therapy (RRT) to correct these critical derangements. The use of RRT is frequently complicated by RRT circuit coagulation. AKI in the ICU mostly occurs due to sepsis, which is a life-threatening condition that arises when the body's response to an infection injures it's own tissues and organs. AKI was also frequently seen in COVID-19. Mechanisms leading to AKI are unclear in both sepsis and COVID-19. This PhD thesis illustrates that in ICU patients, renal function cannot reliably be assessed by blood creatinine due to muscle mass loss. Technical adjustments in RRT resulted in considerably less rapid RRT circuit coagulation. During the COVID-19 pandemic very rapid circuit coagulation was found, which we could substantially delay by the use of the combination of citrate and heparin for RRT circuit anticoagulation. Renal biopsies obtained after dead from patients with sepsis or COVID-19 indicated that the pathophysiological mechanisms for AKI in both entities might be different. In 93% of patients who died from COVID-19 chronic kidney disease (CKD) was found in biopsies indicating CKD is an important risk factor for a severe course of COVID-19

A comparative study of Tam3 and Ac transposition in transgenic tobacco and petunia plants

Transposition of the Anthirrinum majus Tam3 element and the Zea mays Ac element has been monitored in petunia and tobacco plants. Plant vectors were constructed with the transposable elements cloned into the leader sequence of a marker gene. Agrobacterium tumefaciens-mediated leaf disc transformation was used to introduce the transposable element constructs into plant cells. In transgenic plants, excision of the transposable element restores gene expression and results in a clearly distinguishable phenotype. Based on restored expression of the hygromycin phosphotransferase II (HPTII) gene, we established that Tam3 excises in 30% of the transformed petunia plants and in 60% of the transformed tobacco plants. Ac excises from the HPTII gene with comparable frequencies (30%) in both plant species. When the β-glucuronidase (GUS) gene was used to detect transposition of Tam3, a significantly lower excision frequency (13%) was found in both plant species. It could be shown that deletion of parts of the transposable elements Tam3 and Ac, removing either one of the terminal inverted repeats (TIR) or part of the presumptive transposase coding region, abolished the excision from the marker genes. This demonstrates that excision of the transposable element Tam3 in heterologous plant species, as documented for the autonomous element Ac, also depends on both properties. Southern blot hybridization shows the expected excision pattern and the reintegration of Tam3 and Ac elements into the genome of tobacco plants.