Reversible Glutamate Coordination to High-Valent Nickel Protects the Active Site of a [NiFe] Hydrogenase from Oxygen.

NAD+-reducing [NiFe] hydrogenases are valuable biocatalysts for H2-based energy conversion and the regeneration of nucleotide cofactors. While most hydrogenases are sensitive toward O2 and elevated temperatures, the soluble NAD+-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus (HtSH) is O2-tolerant and thermostable. Thus, it represents a promising candidate for biotechnological applications. Here, we have investigated the catalytic activity and active-site structure of native HtSH and variants in which a glutamate residue in the active-site cavity was replaced by glutamine, alanine, and aspartate. Our biochemical, spectroscopic, and theoretical studies reveal that at least two active-site states of oxidized HtSH feature an unusual architecture in which the glutamate acts as a terminal ligand of the active-site nickel. This observation demonstrates that crystallographically observed glutamate coordination represents a native feature of the enzyme. One of these states is diamagnetic and characterized by a very high stretching frequency of an iron-bound active-site CO ligand. Supported by density-functional-theory calculations, we identify this state as a high-valent species with a biologically unprecedented formal Ni(IV) ground state. Detailed insights into its structure and dynamics were obtained by ultrafast and two-dimensional infrared spectroscopy, demonstrating that it represents a conformationally strained state with unusual bond properties. Our data further show that this state is selectively and reversibly formed under oxic conditions, especially upon rapid exposure to high O2 levels. We conclude that the kinetically controlled formation of this six-coordinate high-valent state represents a specific and precisely orchestrated stereoelectronic response toward O2 that could protect the enzyme from oxidative damage.

Binding of exogenous cyanide reveals new active-site states in [FeFe] hydrogenases.

[FeFe] hydrogenases are highly efficient metalloenyzmes for hydrogen conversion. Their active site cofactor (the H-cluster) is composed of a canonical [4Fe-4S] cluster ([4Fe-4S]H) linked to a unique organometallic di-iron subcluster ([2Fe]H). In [2Fe]H the two Fe ions are coordinated by a bridging 2-azapropane-1,3-dithiolate (ADT) ligand, three CO and two CN- ligands, leaving an open coordination site on one Fe where substrates (H2 and H+) as well as inhibitors (e.g. O2, CO, H2S) may bind. Here, we investigate two new active site states that accumulate in [FeFe] hydrogenase variants where the cysteine (Cys) in the proton transfer pathway is mutated to alanine (Ala). Our experimental data, including atomic resolution crystal structures and supported by calculations, suggest that in these two states a third CN- ligand is bound to the apical position of [2Fe]H. These states can be generated both by "cannibalization" of CN- from damaged [2Fe]H subclusters as well as by addition of exogenous CN-. This is the first detailed spectroscopic and computational characterisation of the interaction of exogenous CN- with [FeFe] hydrogenases. Similar CN--bound states can also be generated in wild-type hydrogenases, but do not form as readily as with the Cys to Ala variants. These results highlight how the interaction between the first amino acid in the proton transfer pathway and the active site tunes ligand binding to the open coordination site and affects the electronic structure of the H-cluster.

Factors associated with number of duodenal samples obtained in suspected celiac disease.

BACKGROUND AND STUDY AIMS: Many people with celiac disease are undiagnosed and there is evidence that insufficient duodenal samples may contribute to underdiagnosis. The aims of this study were to investigate whether more samples leads to a greater likelihood of a diagnosis of celiac disease and to elucidate factors that influence the number of samples collected. PATIENTS AND METHODS: We identified patients from two community hospitals who were undergoing duodenal biopsy for indications (as identified by International Classification of Diseases code) compatible with possible celiac disease. Three cohorts were evaluated: no celiac disease (NCD, normal villi), celiac disease (villous atrophy, Marsh score 3), and possible celiac disease (PCD, Marsh score < 3). Endoscopic features, indication, setting, trainee presence, and patient demographic details were evaluated for their role in sample collection. RESULTS: 5997 patients met the inclusion criteria. Patients with a final diagnosis of celiac disease had a median of 4 specimens collected. The percentage of patients diagnosed with celiac disease with one sample was 0.3 % compared with 12.8 % of those with six samples ( P  = 0.001). Patient factors that positively correlated with the number of samples collected were endoscopic features, demographic details, and indication ( P  = 0.001). Endoscopist factors that positively correlated with the number of samples collected were absence of a trainee, pediatric gastroenterologist, and outpatient setting ( P  < 0.001). CONCLUSIONS: Histological diagnosis of celiac disease significantly increased with six samples. Multiple factors influenced whether adequate biopsies were taken. Adherence to guidelines may increase the diagnosis rate of celiac disease.

Repeated use of albuterol inhaler as a potential cause of Takotsubo cardiomyopathy.

PATIENT: Female, 78 FINAL DIAGNOSIS: Takotsubo cardiomyopathy Symptoms: Chest discomfort, chest pain, dyspnea, short of breath Medication: - Clinical Procedure: - Specialty: Cardiology. OBJECTIVE: Unusual or unexpected effect of treatment. BACKGROUND: Takotsubo cardiomyopathy is an increasingly recognized cardiac condition that usually results from an acute stressor. Some medications are becoming recognized as possible stressors. Albuterol is widely used in general medicine. We report an unusual link between Takotsubo cardiomyopathy and albuterol. CASE REPORT: A 78-year-old woman presented to our emergency department for chest pain of 2-day duration. The patient had been taking albuterol inhaler therapy for worsening shortness of breath followed by chest pain. Her albuterol use was excessive. There were no other acute stressors. The electrocardiogram showed ST-elevations in the anterior and inferior leads. Emergent coronary angiography showed noncritical coronary artery disease and left ventriculography showed apical ballooning. CONCLUSIONS: When patients taking albuterol present with acute chest pain in the absence of other etiologies, beta-agonist-induced Takotsubo cardiomyopathy should be considered.