Dataset Information

Catalytic activities of NifEN: implications for nitrogenase evolution and mechanism.

ABSTRACT: NifEN is a key player in the biosynthesis of nitrogenase MoFe protein. It not only shares a considerable degree of sequence homology with the MoFe protein, but also contains clusters that are homologous to those found in the MoFe protein. Here we present an investigation of the catalytic activities of NifEN. Our data show that NifEN is catalytically competent in acetylene (C(2)H(2)) and azide (N(3)(-)) reduction, yet unable to reduce dinitrogen (N(2)) or evolve hydrogen (H(2)). Upon turnover, C(2)H(2) gives rise to an additional S = 1/2 signal, whereas N(3)(-) perturbs the signal originating from the NifEN-associated FeMoco homolog. Combined biochemical and spectroscopic studies reveal that N(3)(-) can act as either an inhibitor or an activator for the binding and/or reduction of C(2)H(2), while carbon monoxide (CO) is a potent inhibitor for the binding and/or reduction of both N(3)(-) and C(2)H(2). Taken together, our results suggest that NifEN is a catalytic homolog of MoFe protein; however, it is only a "skeleton" version of the MoFe protein, as its associated clusters are simpler in structure and less versatile in function, which, in turn, may account for its narrower range of substrates and lower activities of substrate reduction. The resemblance of NifEN to MoFe protein in catalysis points to a plausible, sequential appearance of the two proteins in nitrogenase evolution. More importantly, the discrepancy between the two systems may provide useful insights into nitrogenase mechanism and allow reconstruction of a fully functional nitrogenase from the "skeleton" enzyme, NifEN.

SUBMITTER: Hu Y

PROVIDER: S-EPMC2761346 | biostudies-other | 2009 Oct

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

Catalytic activities of NifEN: implications for nitrogenase evolution and mechanism.

Hu Yilin Y Yoshizawa Janice M JM Fay Aaron W AW Lee Chi Chung CC Wiig Jared A JA Ribbe Markus W MW

Proceedings of the National Academy of Sciences of the United States of America 20090923 40

NifEN is a key player in the biosynthesis of nitrogenase MoFe protein. It not only shares a considerable degree of sequence homology with the MoFe protein, but also contains clusters that are homologous to those found in the MoFe protein. Here we present an investigation of the catalytic activities of NifEN. Our data show that NifEN is catalytically competent in acetylene (C(2)H(2)) and azide (N(3)(-)) reduction, yet unable to reduce dinitrogen (N(2)) or evolve hydrogen (H(2)). Upon turnover, C( ...[more]

PMID: 19805110

Similar Datasets

Project description:Biological nitrogen fixation, the reduction of N(2) to two NH(3) molecules, supports more than half the human population. The predominant form of the enzyme nitrogenase, which catalyzes this reaction, comprises an electron-delivery Fe protein and a catalytic MoFe protein. Although nitrogenase has been studied extensively, the catalytic mechanism has remained unknown. At a minimum, a mechanism must identify and characterize each intermediate formed during catalysis and embed these intermediates within a kinetic framework that explains their dynamic interconversion. The Lowe-Thorneley (LT) model describes nitrogenase kinetics and provides rate constants for transformations among intermediates (denoted E(n), where n is the number of electrons (and protons), that have accumulated within the MoFe protein). Until recently, however, research on purified nitrogenase had not characterized any E(n) state beyond E(0). In this Account, we summarize the recent characterization of three freeze-trapped intermediate states formed during nitrogenase catalysis and place them within the LT kinetic scheme. First we discuss the key E(4) state, which is primed for N(2) binding and reduction and which we refer to as the "Janus intermediate" because it lies halfway through the reaction cycle. This state has accumulated four reducing equivalents stored as two [Fe-H-Fe] bridging hydrides bound to the active-site iron-molybdenum cofactor ([7Fe-9S-Mo-C-homocitrate]; FeMo-co) at its resting oxidation level. The other two trapped intermediates contain reduced forms of N(2). One, intermediate, designated I, has S = 1/2 FeMo-co. Electron nuclear double resonance/hyperfine sublevel correlation (ENDOR/HYSCORE) measurements indicate that I is the final catalytic state, E(8), with NH(3) product bound to FeMo-co at its resting redox level. The other characterized intermediate, designated H, has integer-spin FeMo-co (non-Kramers; S ≥ 2). Electron spin echo envelope modulation (ESEEM) measurements indicate that H contains the [-NH(2)] fragment bound to FeMo-co and therefore corresponds to E(7). These assignments in the context of previous studies imply a pathway in which (i) N(2) binds at E(4) with liberation of H(2), (ii) N(2) is promptly reduced to N(2)H(2), (iii) the two N's are reduced in two steps to form hydrazine-bound FeMo-co, and (iv) two NH(3) are liberated in two further steps of reduction. This proposal identifies nitrogenase as following a "prompt-alternating (P-A)" reaction pathway and unifies the catalytic pathway with the LT kinetic framework. However, the proposal does not incorporate one of the most puzzling aspects of nitrogenase catalysis: obligatory generation of H(2) upon N(2) binding that apparently "wastes" two reducing equivalents and thus 25% of the total energy supplied by the hydrolysis of ATP. Because E(4) stores its four accumulated reducing equivalents as two bridging hydrides, we propose an answer to this puzzle based on the organometallic chemistry of hydrides and dihydrogen. We propose that H(2) release upon N(2) binding involves reductive elimination of two hydrides to yield N(2) bound to doubly reduced FeMo-co. Delivery of the two available electrons and two activating protons yields cofactor-bound diazene, in agreement with the P-A scheme. This keystone completes a draft mechanism for nitrogenase that both organizes the vast body of data on which it is founded and serves as a basis for future experiments.

Project description:The properties and catalytic reactions of the enzyme nitrogenase purified from Klebsiella pneumoniae were studied by electron-paramagnetic-resonance (e.p.r.) spectroscopy at temperatures down to 8 degrees K. The two protein fractions, Kp1 (the iron-molybdenum protein) and Kp2 (the iron protein), were examined alone and in steady-state mixtures and also in pre-steady-state experiments, by using the rapid-freezing method. Kp1 protein in dithionite solution shows a rhombic type of spectrum with g(1) 4.32, g(2) 3.63, g(3) 2.009 at pH6.8 (0 degrees C). Small changes in the spectrum produced by protons (pK=8.7 at 0 degrees C) or by acetylene indicate binding of these oxidizing substrates to this protein fraction. Kp2 protein shows a rhombic spectrum with g(1) 2.053, g(2) 1.942, g(3) 1.865, which integrates to about 0.45 electron/molecule. Binding of ATP, with a dissociation constant of 4x10(-4)m, changes the spectrum to an axial form with g( parallel) 2.036, g( perpendicular) 1.929, thus indicating a conformation change of Kp2 protein. The Kp2 protein spectrum disappears reversibly on cautious oxidation. The signals of both proteins are diminished in their steady-state mixtures, obtained in the presence of ATP and dithionite (with an ATP-generating system and Mg(2+) ions) and with protons, N(2) or acetylene as oxidizing substrate. At the same time as dithionite is consumed in such reactions, the Kp1 protein signal is gradually restored and the Kp2 protein signal diminishes to zero. In rapid-freezing experiments the signals from the two proteins decreased at indistinguishable rates (t((1/2)) about 10ms), then they remained constant. Results are interpreted in terms of a scheme in which reducing equivalents pass from dithionite to Kp2 protein, then, in an ATP-dependent reaction to Kp1 protein, this being finally reoxidized by N(2) or another oxidizing substrate. In this scheme Kp1 protein cycles between its signal-giving state and a very highly reduced signal-free state.

Dataset Information

Catalytic activities of NifEN: implications for nitrogenase evolution and mechanism.

Publications

Catalytic activities of NifEN: implications for nitrogenase evolution and mechanism.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets