A Novel Report on Assessing pH Dependent Role of Nitrate Reductase on Green Biofabrication of Gold Nanoplates and Nanocubes

Denitrification is generally considered to occur under micro-oxic or anoxic conditions. With this in mind, the physiological function and regulation of several steps in the denitrification of model α-proteobacteria are compared in the present review. Expression of the periplasmic nitrate reductase is quite variable, with this enzyme being maximally expressed under oxic conditions in some bacteria, but under micro-oxic conditions in others. Expression of nitrite and NO reductases in most denitrifiers is more tightly controlled, with expression only occurring under micro-oxic conditions. A possible exception to this may be Roseobacter denitrificans, but the physiological role of these enzymes under oxic conditions is uncertain.

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Conformational Destabilization of Immunoglobulin G Increases the Low pH Binding Affinity with the Neonatal Fc Receptor

Journal of Biological Chemistry ◽

10.1074/jbc.m115.691568 ◽

2015 ◽

Vol 291 (4) ◽

pp. 1817-1825 ◽

Cited By ~ 23

Author(s):

Benjamin T. Walters ◽

Pernille F. Jensen ◽

Vincent Larraillet ◽

Kevin Lin ◽

Thomas Patapoff ◽

...

Keyword(s):

Binding Affinity ◽

Hydrogen Exchange ◽

Dissociation Constants ◽

Conformational Dynamics ◽

Fc Receptor ◽

Salt Bridges ◽

Neonatal Fc Receptor ◽

Intermolecular Contacts ◽

Ph Dependent

Crystallographic evidence suggests that the pH-dependent affinity of IgG molecules for the neonatal Fc receptor (FcRn) receptor primarily arises from salt bridges involving IgG histidine residues, resulting in moderate affinity at mildly acidic conditions. However, this view does not explain the diversity in affinity found in IgG variants, such as the YTE mutant (M252Y,S254T,T256E), which increases affinity to FcRn by up to 10×. Here we compare hydrogen exchange measurements at pH 7.0 and pH 5.5 with and without FcRn bound with surface plasmon resonance estimates of dissociation constants and FcRn affinity chromatography. The combination of experimental results demonstrates that differences between an IgG and its cognate YTE mutant vary with their pH-sensitive dynamics prior to binding FcRn. The conformational dynamics of these two molecules are nearly indistinguishable upon binding FcRn. We present evidence that pH-induced destabilization in the CH2/3 domain interface of IgG increases binding affinity by breaking intramolecular H-bonds and increases side-chain adaptability in sites that form intermolecular contacts with FcRn. Our results provide new insights into the mechanism of pH-dependent affinity in IgG-FcRn interactions and exemplify the important and often ignored role of intrinsic conformational dynamics in a protein ligand, to dictate affinity for biologically important receptors.

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Dissociation of FAD from the NAD(P)H-Nitrate Reductase Complex from Ankistrodesmus braunii and Role of Flavin in Catalysis

Zeitschrift für Naturforschung C ◽

10.1515/znc-1982-1-206 ◽

1982 ◽

Vol 37 (1-2) ◽

pp. 24-30 ◽

Cited By ~ 10

Author(s):

Miguel A. De la Rosa ◽

Antonio J. Márquez ◽

José M. Vega

Keyword(s):

Nitrate Reductase ◽

Reductase Activity ◽

Prosthetic Group ◽

Original Activity ◽

Enzyme Preparations ◽

Fluorescence Studies ◽

Transport Chain ◽

Sulphydryl Groups ◽

Nadh Cytochrome

Ankistrodesmus braunii NAD(P)H-nitrate reductase is a complex hemoflavomolybdoprotein composed by eight similar subunits. The flavin prosthetic group, identified as FAD, is essential for the NAD(P)H-dependent activities of the complex, and is located before the heme chromo- phore in the enzyme electron transport chain from reduced pyridine nucleotides to nitrate. Fluorescence studies indicate that nitrate reductase can dissociate about 80% of its FAD by incubation at room temperature, the flavin dissociation being followed by a parallel decrease of NADH-nitrate reductase activity. Dissociation of FAD from the protein is easily increased by dilution or prolonged dialysis of the enzyme preparations. However, exogenous FAD specifically prevents the dissociation of enzyme-bound flavin, and protects the NAD(P)H-dependent activities. The Km for FAD, as a protector of NADH-cytochrome c reductase activity, is 4 nᴍ. In addition, dithioerythritol also prevents the flavin dissociation, and therefore the presence of free sulphydryl groups in the FAD-domain is suggested. FAD-depleted nitrate reductase, obtained by several methods, is unable to recover its original activity when incubated in the presence of FAD alone or with thiols.

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