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Nitrogenase of Klebsiella pneumoniae. Reversibility of the reductant-independent MgATP-cleavage reaction is shown by MgADP-catalysed phosphate/water oxygen exchange.

ABSTRACT: The steady-state kinetics of reductant-independent ATP hydrolysis by Klebsiella pneumoniae nitrogenase at 23 degrees C at pH 7.4 were determined as a function of component protein ratio (optimal at an oxidized Fe protein/MoFe protein ratio of 3:1) and MgATP concentration (Km 400 microM). Competitive inhibition was observed for MgADP (Ki 145 microM), [beta gamma-methylene]ATP (Mgp[CH2]ppA) (Ki 115 microM), [beta gamma-monofluoromethylene]ATP (Mgp[CHF]ppA) (Ki 53 microM) and [beta gamma-difluoromethylene]ATP (Mgp[CF2]ppA) (Ki 160 microM). The tighter binding of MgADP to free oxidized Fe protein (KD less than 10 microM) than to the oxidized Fe protein-MoFe protein complex (Ki 145 microM) is proposed as the driving force that induces rate-limiting protein dissociation in the catalytic cycle of nitrogenase. The reversible nature of the reductant-independent MgATP-cleavage reaction was demonstrated by an MgADP-induced enhancement of the rate of the phosphate/water oxygen exchange reaction with 18O-labelled phosphate ion. This enhancement, like the reductant-independent ATPase reaction, only occurred with the complex formed by oxidized Fe protein and MoFe protein and not with the individual proteins. The results are discussed in terms of the mechanism of ATP hydrolysis by nitrogenase and other systems involving protein-protein interactions.

SUBMITTER: Thorneley RN

PROVIDER: S-EPMC1151306 | biostudies-other | 1991 Aug

REPOSITORIES: biostudies-other

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Project description:1. Nitrogenase from the facultative anaerobe Klebsiella pneumoniae was resolved into two protein components resembling those obtained from other nitrogen-fixing bacteria. 2. Both proteins were purified to homogeneity as shown by the criteria of disc electrophoresis and ultracentrifugal analysis. 3. The larger component had a mol.wt. of 218000 and contained one Mo atom, 17Fe atoms and 17 acid-labile sulphide groups/mol; it contained two types of subunit, present in equal amounts, of mol.wts. 50000 and 60000. All the common amino acids were present, with a predominance of acidic residues. The apparent partial specific volume was 0.73; ultracentrifugal analysis gave s(0) (20,w)=11.0S and D(0) (20,w)=4.94x10(-7)cm(2)/s. The specific activities (nmol of product formed/min per mg of protein) when assayed with the second nitrogenase component were 1500 for H(2) evolution, 380 for N(2) reduction, 1200 for acetylene reduction and 5400 for ATP hydrolysis. The reduced protein showed electron-paramagnetic-resonance signals at g=4.3, 3.7 and 2.015; the Mössbauer spectrum of the reduced protein consisted of at least three doublets. The u.v. spectra of the oxidized and reduced proteins were identical. On oxidation the absorbance increased generally throughout the visible region and a shoulder at 430nm appeared. The circular-dichroism spectra of both the oxidized and reduced proteins were the same, consisting mainly of a negative trough at 220nm. 4. The smaller component had mol.wt. 66800 and contained four Fe atoms and four acid-labile sulphide groups in a molecule comprising two subunits each of mol.wt. 34600. All common amino acids except tryptophan were present, with a predominance of acidic residues. The apparent partial specific volume calculated from the amino acid analysis was 0.732, which was significantly higher than that obtained from density measurements (0.69); ultracentrifugal analysis gave s(0) (20,w)=4.8S and D(0) (20,w)=5.55x10(-7)cm(2)/s. The specific activities (nmol of product formed/min per mg of protein) were 1050 for H(2) evolution, 275 for N(2) reduction, 980 for acetylene reduction and 4350 for ATP hydrolysis. The protein was not cold-labile. The reduced protein showed electron-paramagnetic-resonance signals in the g=1.94 region. The Mössbauer spectrum of the reduced protein consisted of a doublet at 77 degrees K. The u.v. spectra of reduced and O(2)-inactivated proteins were identical, and inactivation by O(2) generally increased the absorbance in the visible region and resulted in a shoulder at 460nm. The circular-dichroism spectra exhibited a negative trough at 220nm and inactivation by O(2) decreased the depth of the trough. 5. The reduction of N(2) and acetylene, and H(2) evolution, were maximal at a 1:1 molar ratio of the Fe-containing protein to the Mo-Fe-containing protein; excess of the Mo-Fe-containing protein was inhibitory. All reductions were accompanied by H(2) evolution. The combined proteins had no ATP-independent hydrogenase activity.

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Nitrogenase of Klebsiella pneumoniae. Reversibility of the reductant-independent MgATP-cleavage reaction is shown by MgADP-catalysed phosphate/water oxygen exchange.

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