Hydrolysis of Nucleoside Triphosphates Other than ATP by Nitrogenase

Abstract

The hydrolysis of ATP to ADP and P i is an integral part of all substrate reduction reactions catalyzed by nitrogenase. In this work, evidence is presented that nitrogenases isolated from Azotobacter vinelandii and Clostridium pasteurianum can hydrolyze MgGTP, MgITP, and MgUTP to their respective nucleoside diphosphates at rates comparable to those measured for MgATP hydrolysis. The reactions were dependent on the presence of both the iron ( The hydrolysis of ATP to ADP and P i has long been known to be an integral part of all nitrogenase substrate reduction reactions (1-4). The stoichiometry for coupling ATP hydrolysis to substrate reduction varies depending on reactions conditions, with a minimum of 2 ATP molecules hydrolyzed for each electron transferred to substrate (5, 6). Thus, the reduction of N 2 to 2NH 3 by nitrogenase requires the hydrolysis of at least 16 ATP molecules (Equation 1). There have been isolated examinations of the interactions of nucleotides other than adenine nucleotides with nitrogenase. To summarize, no nucleotide other than ATP has been shown to support substrate reduction, although there is a recent report that protons can be reduced at extremely low rates without ATP within an Fe protein-MoFe protein complex created with AlF 4 Ϫ ⅐ADP (10). The hydrolysis of ATP to ADP and P i is the predominant reaction observed. Apparently, the MoFe protein alone or the Fe protein-MoFe protein complex can catalyze the further hydrolysis of ADP at extremely low rates (11,12), although these reactions do not appear to be part of the substrate reduction mechanism. A divalent metal-bound form of ATP is required for hydrolysis, with MgATP showing the highest rates of substrate reduction (13). There is, however, evidence suggesting that nucleotides other than adenosine di-or triphosphates interact with nitrogenase. The Fe protein is known to bind 2 molecules of either MgATP or MgADP, with each resulting in distinct protein conformational changes (1). These protein conformational changes have been detected by a variety of methods. For example, the line shape of the EPR signal of the S ϭ 1/2 state of the reduced 1ϩ cluster of the Fe protein changes upon MgATP or MgADP binding (14). MgGTP was observed to induce small changes in the EPR line shape of the Fe protein, suggesting that MgGTP binds to the Fe protein and induces at least partial protein conformational changes (14). Another technique that reports protein conformational changes in the Fe protein is the sensitivity of the [4Fe-4S] cluster to chelation by iron-specific chelators like bathophenanthroline or ␣,␣Ј-dipyridyl. In the absence of nucleotides, the [4Fe-4S] cluster of the Fe protein is resistant to chelation by chelators (15, 16). Upon binding MgATP, however, a timedependent release of all of the iron atoms from the cluster to

Purification and Characterization of Membrane-associated CooC Protein and Its Functional Role in the Insertion of Nickel into Carbon Monoxide Dehydrogenase from Rhodospirillum rubrum

“…Purified CooC is a homodimer and has NTPase activities. These properties are similar to those of A. vinelandii NifH (nitrogen fixation) (37,40) or E. coli HypB (nickel-processing for hydrogenase) (41) which are also homodimers with NTPase activities; CooC has sequence similarity to both of these proteins. However, CooC and NifH reveal a higher ATPase/GTPase ratio compared with E. coli HypB.…”

Conformational Re‐engineering of Porphyrins as Receptors with Switchable N−H⋅⋅⋅X‐Type Binding Modes

“…Many cofactors,enzyme substrates,and DNAare anionic in nature. [13] Among anions of current interest, [14] the pyrophosphate anion has attracted particular attention due to its biological relevance, [15] and significant efforts have been made to develop more potent pyrophosphate sensors. [16] To study the binding capabilities of the isolated tetraaminoporphyrin a 4 -1,U V/Vis titrations were carried out with tris(tetrabutylammonium) hydrogen pyrophosphate (PP i )inthe presence of TFA.…”

Konformativer Umbau von Porphyrinen als Rezeptoren mit schaltbaren N‐H⋅⋅⋅X‐Bindungsmodi

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Die Selektivitätu nd funktionale Variabilitätv on Porphyrinkofaktoren basiert typischerweise auf der Substratbindung durchM etalloporphyrine,w obei die Pyrrolstickstoffatome nur zur Chelatisierung der Metallionen dienen. In einem ersten Schritt zu Porphyrinzentren mit "enzymähnlicher" Aktivitätz eigt eine strukturelle und spektroskopische Untersuchung der Substratbindung im Kern jedoch, dass ein sattelverbogenes Porphyrin mit peripheren Aminorezeptorgruppen (1,2 ,3,7,8,12,13,17,10,15,20-tetrakis(2-aminophenyl)porphyrin), abhängig von der Aziditätder Lçsung,A nalyte in einer schaltbaren Weise koordiniert. Das supramolekulare Ensemble weist eine hohe Affinitätu nd Selektivitätf ürd as Pyrophosphatanion (2.26 AE 0.021) 10 9 m À1 auf.

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“…Eine modifizierte Lindsey-Kondensation von 3,4-Diethylpyrrol [12] Viele Kofaktoren, Enzymsubstrate und DNAs ind anionischer Natur. [13] Unter den gegenwärtig im Interesse stehenden Anionen [14] hat das Pyrophosphatanion aufgrund seiner biologischen Relevanz [15] besondere Aufmerksamkeit erregt. Es wurden erhebliche Anstrengungen unternommen, um leistungsfähigere Pyrophosphatsensoren zu entwickeln.…”