Mechanistic Study of Heme Protein-Mediated Nitric Oxide Dioxygenation Using Photolytically Produced Nitric Oxide

The previously reported NO precursor [Mn(PaPy2Q)(NO)]ClO4 (1), where (PaPy2QH) is N,N-bis(2-pyridylmethyl)-amine-N-ethyl-2-quinoline-2-carboxamide, was synthesized and proven capable of producing as much as 180 µM NO when irradiated by a single 3 mJ 500nm laser pulse, in a 0.15 cm path cell, without the need for additional sacrificial reductants or oxidants. Species 1 was first used to study the reaction of nitric oxide with oxy-myoglobin (oxyMb) to form ferric myoglobin (metMb) and nitrate. This reaction had long been assumed to proceed via the same iron-bound peroxynitrite intermediate (metMb(OONO)) as the metMb-catalyzed isomerization of peroxynitrite to nitrate. Recent research showed that the metMb-catalyzed isomerization of peroxynitrite to nitrate produces detectable amounts of nitrogen dioxide and ferryl myoglobin (ferrylMb). This suggested a “caged NO2” mechanism for peroxynitrite isomerization. The presence of free NO2 and ferrylMb products revealed that small amounts of NO2 escape from myoglobin’s interior before recombination can occur, and these should also be generated in the reaction of oxyMb with NO, if the common intermediate metMb(OONO) is formed. However, in time resolved UV/Vis spectroscopy experiments reported herein no ferrylMb was detected when oxyMb and NO reacted. The sensitivity of the methodology is such that as little as 10% of the ferrylMb predicted from the experiments with metMb and peroxynitrite should have been detectable. These results lead to the conclusion that the oxyMb + NO and metMb + ONOO− reactions do not proceed via a common intermediate as previously thought. The conclusion has significant implications for researchers that propose a possible role of oxyMb in intracellular NO regulation, because toxic NO2 and ferrylMb are not generated during NO oxidation by this species. Nitric oxide precursor 1 was then used for investigating the interaction between NO and the protein truncated hemoglobin N (trHbN) from the pathogen Mycobacterium tuberculosis. Oxy-trHbN is exceptionally efficient at converting NO to nitrate, with a reported rate constant of 7.45108 M1 s1 compared to 4107 M1 s1 for oxyMb. This work analyzed the NO dioxygenation kinetics of wild type trHbN and a set of variants, as well as the nitrosylation kinetics for the reduced (red-trHbN) forms of these proteins. The NO dioxygenation reaction was remarkably insensitive to mutations, even within the active site, while nitrosylation was somewhat more sensitive. Curiously, the most profound change to the rate constant for nitrosylation was effected by deletion of a 12 amino acid N-terminal sequence. The deletion mutant exhibited first-order kinetics with respect to NO, but was zero-order with respect to protein concentration; by contrast all other variants exhibited second-order rate constants greater than 108 M1s1. TrHbN boasts an extensive tunnel system that connects the protein exterior with the active site, and is likely the main contributor to the protein’s impressive NO dioxygenation efficiency. The results herein suggest that N-terminal deletion abolishes a large-scale conformational motion, in the absence of which NO can still readily enter the tunnel system, but is prevented from binding to the heme for an extended period of time

PHYSICAL ACTIVITY, SLEEP PATTERNS, AND HEALTH OUTCOMES IN UNIVERSITY LAW ENFORCEMENT OFFICERS

Research indicates that law enforcement officers (LEOs) have a higher prevalence of developing coronary artery disease (CAD) compared to the general population. Sleep deprivation and physical inactivity have been found to be related to many risk factors for CAD. This cross-sectional study examined the health status and the relationship between sleep and physical activity outcomes versus CAD risk factors among 27 University LEOs. The subjects’ health behaviors, and CAD and metabolic syndrome risk factors were described using basic statistics. Accelerometer derived sleep and physical activity outcomes were correlated to measures of health to identify potential relationships. 33% of LEOs were classified as moderate risk for CAD; 92% had dyslipidemia, 58% had elevated triglycerides, 23% had prediabetes, and 22% suffered from obesity. The administrators and first shift LEOs slept more compared to second or third shift LEOs. The LEOs were more sedentary while on-duty. In addition, sedentary time was correlated to systolic blood pressure. LEOs accumulated 24.4 min·d-1 of moderate-to-vigorous physical activity (MVPA), but only spent 9.3 min·d-1 in continuous bouts of MVPA. In conclusion, multiple CAD risk factors were present in these LEOs and achieving adequate amounts of physical activity and sleep may decrease their risk of developing chronic diseases

A case of a supernumerary third head of the biceps brachii muscle - clinical significance

A three-headed biceps brachii muscle was found in one male cadaver out of 118 (0.85%) studied, both for educational and research purposes. The supernumerary head of the biceps brachii muscle was located superficially to the normal heads. It was originated from the insertion of the pectoralis major tendon at humerus. At the lower third of the arm, it was merging with the normal biceps brachii muscle and as a conjoint tendon was inserted at the radial tuberosity. The significance of our finding lies on the fact that the location of the supernumerary head was superficial to the other two heads, in contrast to previous reports in the literature. Surgeons and especially orthopaedic surgeons should bear in mind muscular variations like the one reported in the present study

The cadaver of a Caucasian man with a supernumerary fourth dorsal interosseous muscle in the right hand: a case report

<p>Abstract</p> <p>Introduction</p> <p>The human hand is a complex anatomic entity consisting of many muscles, nerves, and vessels, thus providing a special ability to perform accurate and meticulous movements. In this group of muscles are the four dorsal interosseous muscles.</p> <p>Case presentation</p> <p>A distinct supernumerary fourth dorsal interosseous muscle was found in the right hand of the cadaver of a 76-year-old Caucasian man without any other concomitant abnormality.</p> <p>Conclusions</p> <p>The presence of such an additional muscle in the hand should be considered in the management of hand deformities, whether the treatment is conservative or surgical.</p

Modifying the Steric Properties in the Second Coordination Sphere of Designed Peptides Leads to Enhancement of Nitrite Reductase Activity

Protein design is a useful strategy to interrogate the protein structureâ function relationship. We demonstrate using a highly modular 3â stranded coiled coil (TRIâ peptide system) that a functional typeâ 2 copper center exhibiting copper nitrite reductase (NiR) activity exhibits the highest homogeneous catalytic efficiency under aqueous conditions for the reduction of nitrite to NO and H2O. Modification of the amino acids in the second coordination sphere of the copper center increases the nitrite reductase activity up to 75â fold compared to previously reported systems. We find also that steric bulk can be used to enforce a threeâ coordinate CuI in a site, which tends toward twoâ coordination with decreased steric bulk. This study demonstrates the importance of the second coordination sphere environment both for controlling metalâ center ligation and enhancing the catalytic efficiency of metalloenzymes and their analogues.Erstklassiges aus der zweiten Reihe: Die Aktivität der Nitritreduktase kann durch Modifikation der sterischen Eigenschaften in der zweiten Koordinationssphäre eines Typâ 2â Kupferzentrums deutlich erhöht werden. à ber die Sterik lassen sich die Koordination und Reaktivität des Metalls in einem dreisträngigen â Coiledâ coilâ â TRIâ Peptidgerüst (TRIWâ H) vorgeben.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142882/1/ange201712757_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142882/2/ange201712757-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142882/3/ange201712757.pd

Modifying the Steric Properties in the Second Coordination Sphere of Designed Peptides Leads to Enhancement of Nitrite Reductase Activity

Protein design is a useful strategy to interrogate the protein structureâ function relationship. We demonstrate using a highly modular 3â stranded coiled coil (TRIâ peptide system) that a functional typeâ 2 copper center exhibiting copper nitrite reductase (NiR) activity exhibits the highest homogeneous catalytic efficiency under aqueous conditions for the reduction of nitrite to NO and H2O. Modification of the amino acids in the second coordination sphere of the copper center increases the nitrite reductase activity up to 75â fold compared to previously reported systems. We find also that steric bulk can be used to enforce a threeâ coordinate CuI in a site, which tends toward twoâ coordination with decreased steric bulk. This study demonstrates the importance of the second coordination sphere environment both for controlling metalâ center ligation and enhancing the catalytic efficiency of metalloenzymes and their analogues.Second is best: A significant increase in nitrite reductase activity is achieved by modification of the steric properties of the second coordination sphere of a typeâ 2 copper center. The steric properties can be harnessed to control metal coordination and reactivity in a 3â stranded coiled coil TRI peptide scaffold (TRIWâ H).Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142897/1/anie201712757.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142897/2/anie201712757-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142897/3/anie201712757_am.pd

Making or Breaking Metal- Dependent Catalytic Activity: The Role of Stammers in Designed Three- Stranded Coiled Coils

While many life- critical reactions would be infeasibly slow without metal cofactors, a detailed understanding of how protein structure can influence catalytic activity remains elusive. Using de novo designed three- stranded coiled coils (TRI and Grand peptides formed using a heptad repeat approach), we examine how the insertion of a three residue discontinuity, known as a stammer insert, directly adjacent to a (His)3 metal binding site alters catalytic activity. The stammer, which locally alters the twist of the helix, significantly increases copper- catalyzed nitrite reductase activity (CuNiR). In contrast, the well- established zinc- catalyzed carbonic anhydrase activity (p- nitrophenyl acetate, pNPA) is effectively ablated. This study illustrates how the perturbation of the protein sequence using non- coordinating and non- acid base residues in the helical core can perturb metalloenzyme activity through the simple expedient of modifying the helical pitch adjacent to the catalytic center.The addition of a stammer discontinuity within a de novo designed 3SCC containing a symmetric (His)3 metal binding site enhances copper nitrite reductase activity and ablates zinc esterase activity. These results suggest catalytic activity of designed α- helical systems can be modulated by inclusion of discontinuity insertions and deletions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/163447/3/anie202008356-sup-0001-misc_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163447/2/anie202008356_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163447/1/anie202008356.pd

Katalyse und Elektronentransfer in helikalen De‐novo‐Gerüststrukturen

Die Frage nach der Beziehung zwischen der Struktur und der Funktion von Proteinen ist eines der größten Rätsel der Biochemie. Das De‐novo‐Design von Metalloproteinen bietet die Möglichkeit, neu zu bestimmen, was nötig ist, um von Grund auf Funktionalität in einer Struktur aufzubauen, die nicht von der Struktur des natürlichen Vorbilds abgeleitet ist. Dieser Aufsatz konzentriert sich auf Arbeiten zum Proteindesign, die De‐novo‐Metalloproteine innerhalb alpha‐helikaler Gerüststrukturen liefern. Beispiele umfassen De‐novo‐Proteine mit Carboanhydrase‐ oder Nitritreduktaseaktivität, sowie Systeme, in denen die spektroskopischen Eigenschaften einzigartiger Elektronentransferzentren von Cupredoxinen oder Rubredoxinen nachgebildet werden. Diese Arbeiten demonstrieren die Vielseitigkeit von alpha‐Helices als Gerüststrukturen im Design von Metalloproteinen und die Fortschritte, die durch rationales Design möglich sind. Unsere Arbeiten belegen die Unabhängigkeit der Carboanhydraseaktivität von der Gerüststruktur und Position des aktiven Zentrums, verfeinern unsere Cupredoxinmodelle und erhöhen die Aktivität unserer Nitritreduktase um bis zu das 1000‐Fache.Durch De‐novo‐Design von Metalloproteinen lässt sich bestimmen, was nötig ist, um von Grund auf Funktionalität in einer Struktur aufzubauen, die nicht vom natürlichen Vorbild abgeleitet ist. Dieser Aufsatz diskutiert Proteindesign, das De‐novo‐Metalloproteine innerhalb alpha‐helikaler Gerüste liefert; er zeigt zudem die Vielseitigkeit von alpha‐Helices als Gerüste beim Metalloproteindesign sowie die Fortschritte, die durch rationales Design möglich sind.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155507/1/ange201907502_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155507/2/ange201907502.pd

Making or Breaking Metal- Dependent Catalytic Activity: The Role of Stammers in Designed Three- Stranded Coiled Coils

While many life- critical reactions would be infeasibly slow without metal cofactors, a detailed understanding of how protein structure can influence catalytic activity remains elusive. Using de novo designed three- stranded coiled coils (TRI and Grand peptides formed using a heptad repeat approach), we examine how the insertion of a three residue discontinuity, known as a stammer insert, directly adjacent to a (His)3 metal binding site alters catalytic activity. The stammer, which locally alters the twist of the helix, significantly increases copper- catalyzed nitrite reductase activity (CuNiR). In contrast, the well- established zinc- catalyzed carbonic anhydrase activity (p- nitrophenyl acetate, pNPA) is effectively ablated. This study illustrates how the perturbation of the protein sequence using non- coordinating and non- acid base residues in the helical core can perturb metalloenzyme activity through the simple expedient of modifying the helical pitch adjacent to the catalytic center.The addition of a stammer discontinuity within a de novo designed three- stranded coiled coil containing a symmetric (His)3 metal binding site enhances copper nitrite reductase activity and ablates zinc esterase activity. These results suggest catalytic activity of designed α- helical systems can be modulated by inclusion of discontinuity insertions and deletions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/163477/3/ange202008356_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163477/2/ange202008356-sup-0001-misc_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163477/1/ange202008356.pd

The BEACH domain containing protein SPIRRIG in an interplay with TZF proteins and the characterization of SPIRRIG in M. polymorpha

The BEACH domain containing proteins (BDCPs) represent a highly conserved family among eukaryotes and members have been described in most of the prominent model organisms. The BDCPs were discovered through exploration of a severe human disease comprising symptoms such as albinism, bleeding diathesis, immunodeficiency and cancer growth. On the cellular level, BDCPs are classically known to be involved in membrane trafficking processes including vesicle transport, autophagy as well as membrane fission and fusion events. In accordance, the Arabidopsis thaliana cell morphogenesis and BDCP-coding gene SPIRRIG is implicated in membrane-dependent processes as well. The protein is a particularly interesting BDCP, as to date, it is the only member of this family known to exert an additional function in mRNA metabolism by mediating mRNA stability and promoting processing body formation biologically relevant to A. thaliana salt stress response. The exact molecular mechanism of SPIRRIG, however, remains elusive. This study was set out to investigate SPIRRIG function using two different approaches. One part of this work aimed to gain further understanding of SPIRRIG’s molecular function in salt stress response by analyzing an interplay with tandem zinc finger (TZF) proteins. While molecular functions of plant TZF proteins are not yet fully understood, mammalian homologs are involved in stress-associated mRNA destabilization processes upon binding to specific elements in 3’UTRs of target mRNAs. An interaction of SPIRRIG with TZF10 strongly suggested a potential connection of the two proteins and prompted further investigations in order to unravel this so far unknown interplay between BDCPs and TZFs. Strikingly, this study confirmed TZF10 to rescue the salt hypersensitivity phenotype of spirrig mutants. Extensive mRNA expression and stability assays however could not confirm previously hypothesized, biologically relevant roles of the TZF10 3’UTR in the context of spirrig salt hypersensitivity rescue. Nonetheless, this work convincingly demonstrated general mRNA destabilization capacity mediated by the TZF10 3’UTR and isolated a potential mRNA destabilization-inducing motif. The second part of this work utilized an evolutionarily comparative approach in order to unravel conserved, basal BDCP functions in one of the first plants colonizing the land, the liverwort Marchantia polymorpha. The characterization on the morphological and cellular level of a Mpspirrig mutant, which was identified in a T-DNA screen by its short rhizoids, revealed exciting insights on potential BDCP function in the ancient plant. The Mpspirrig short rhizoid phenotype, reminiscent to the short root hair phenotype of Atspirrig, was surprisingly shown to be an expression of a phenotype hallmarked by an increased number of at the same time significantly smaller cells in Mpspirrig gemmae. This newly discovered phenotype pointed to a role of MpSPIRRIG in cell proliferation and/or cell expansion processes. Moreover, this work strongly supported a connection of MpSPIRRIG to membrane trafficking pathways by demonstrating direct co-localization and interaction of MpSPIRRIG with endosomal sorting complex required for transport (ESCRT) components. Finally, this study revealed MpSPIRRIG to be relevant to Marchantia salt stress response and to locate to cytoplasmic mRNA granules, providing several indications for the dual function of SPIRRIG in the two assumingly unrelated pathways of salt stress response and membrane trafficking to be evolutionarily conserved