David J. Lowe

Nitrogenase of klebsiella pneumoniae: A pre-steady state burst of ATP hydrolysis is coupled to electron transfer between the component proteins

Nitrogenase, the enzyme responsible for biological nitrogen flucation is comprised of two redox proteins, both of which contain Fe-S clusters and one of which, the MO-Fe protein, additionally contains MO. Substrate reduction requires both proteins and is obligatorily coupled to the hydrolysis of MgATP to MgADP and Pi [ 1,2] . Pre-steady state EPR [3,4] and stopped flow spectrophotometric studies [.5&j together with M~ssbauer studies [‘7f have shown that MgATP is required for the rapid transfer of electrons from the Fe protein to the MO-Fe protein. Binding of MgATP to the Fe protein results in a lowering of its redox potential [S,9] from -290 to -400 mV [8] . The question arises whether MgATP acts merely as an activator of the Fe protein, allowing electron transfer to the MO-Fe protein, or whether MgATP hydrolysis is coupled to this electron transfer. Kinetic data presented here demonstrate a pre-steady state burst of MgATP hydrolysis which occurs with the same time constant as does electron transfer between the nitrogenase proteins. These observations, and their implications for the role of MgATP are discussed.

Nitrogenase of Azotobacter chroococcum. Kinetics of the reduction of oxidized iron-protein by sodium dithionite

The kinetics of the reduction of oxidized Fe-protein of nitrogenase from Azotobacter chroococcum by sodium dithionite were studied by stopped-flow and rapid-freezing e.p.r. (electron-paramagnetic-resonance) spectroscopy. The appearance of the gav. = 1.94 e.p.r. signal (0.24 electron integrated intensity/mol) was associated with a one-electron reduction by SO2--with k greater than 10(8)M-1-S-1 at 23 degrees C. A value of k = 1.75s-1 was obtained for the rate of dissociation of S2O42- into 2SO2-- at 23 degrees C. Further reductions by SO2-- occurred in three slower phases with rate constants in the range 10(4) -10(6)M-1-S-1. These latter phases have no corresponding e.p.r. signal changes and are probably associated with enzymically inactive protein. The high rate of reduction by SO2-- of the Fe-protein alone (k greater than 10(8)M-1-S-1) relative to the rate of oxidation of the Fe-protein in the catalytically active Fe:Mo-Fe protein complex (k = 2.2 X 1O(2)s-1) and the observation that in the steady state the Fe-protein is substantially oxidized means that at normal assay concentrations another reaction must limit the rate of reduction of Fe-protein during turnover.

Mesencephalic and diencephalic cobalt-lysine injections in an elasmobranch: evidence for two parallel electrosensory pathways.

Iontophoretic injections of cobaltous lysine have revealed a direct projection from the electroreceptive lateral mesencephalic nucleus to the ipsilateral diencephalic posterior lateral nucleus in the thornback ray, Platyrhinoidis triseriata. Since it is known that the latter nucleus projects to the contralateral pallium, this completes the anatomical description of an electrosensory pathway from receptors to telencephalon in an elasmobranch. In addition, diencephalic injections into the electroreceptive anterior nucleus indicate there is a second, parallel electrosensory system which projects to the hypothalamus.

Nitrogenase of Azotobacter chroococcum: Inhibition by ADP of the reduction of oxidised Fe protein by sodium dithionite

Nitrogenase comprises two (non-haem) i ronsulphur proteins, one of which contains molybdenum. Both proteins, an electron donor (ferredoxin, flavodoxin, Na2S204), Mg-ATP and an anaerobic environment are necessary to reduce substrate; Mg-ADP is an inhibitor. During enzyme turnover electrons are transferred in the direction: donor I ~ Fe protein II Mo-Fe protein ~ reducible substrate. Spectrephotometric and rapid freeze e.p.r.* studies on the complete enzymically active nitrogenase system indicates that reactions I and III are slow compared with reaction II [1 -5 ] . The nitrogenase system is extremely complex and, therefore, the study of partial reactions involving individual component proteins has contributed to the understanding of the enzyme mechanism. There is good evidence that both ATP and ADP associate with and induce conformational changes in the reduced Fe protein [4,6-8] and that ATP-dependent electron transfer occurs between the reduced Fe protein and oxidised Mo-Fe protein (reaction II,) [1 -5 ] . There have been no detailed kinetic studies on reaction I of the above sequence, possibly because the nitro-

The Iron-Molybdenum Cofactor of Nitrogenase

It is now eight years since Shah, Brill (1977) published their method for isolating the iron-molybdenum cofactor (FeMoco) from the MoFe protein. Their results indicated that the Mo and Fe were combined with inorganic sulphide in an unique extractable cluster that could activate extracts of certain mutants lacking MoFe protein activity. There had long been a prejudice, based on circumstantial evidence (Smith, 1977), that Mo was at the substrate binding site of nitrogenase and it was assumed that FeMoco was this site. Since that seminal paper a number of groups have studied FeMoco and there is now better (but not absolutely conclusive) evidence on its function. However its structure is not known, its composition is not well defined and its biosynthesis has proved extremely complex. In this paper these topics are reviewed and recent experiments on them described.

Electron-electron double resonance measurements on xanthine oxidase

Electron-electron double resonance measurements were carried out on milk xanthine oxidase (xanthine:oxygen oxidoreductase EC 1.2.3.2) and the spectra obtained supported a previous model, based on EPR data, proposing a spin-spin interaction between unpaired electrons associated with Fe-S and Mo. The technique demonstrated that the additional apparently isotropic, splitting in the Mo EPR spectra observed at low temperature is produced by a single site giving two spectra interconverting at a rate consistent with the Fe-S spin lattice relaxation time. Other data concerning the model and the relaxation behaviour of the species are discussed.

Stereospecific protonation of coordinated alkynes

Abstract The reaction of [V( η 5 -C 5 H 5 ) 2 ( η 2 -PhCCPh)] with anhydrous HCl in tetrahydrofuran gives [V( η 5 -C 5 H 5 ) 2 Cl 2 ] together with cis -PhCHCHPh (ca. 85%) and trans -PhCHCHPh (ca. 15%). Kinetic studies indicate that the formation of cis -PhCHCHPh involves initial protonation of the metal followed by migration of the hydride on to the alkyne to give the cis -vinyl species. Subsequent further protonation gives cis -PhCHCHPh. trans -PhCHCHPh is formed by direct protonation of the coordinated alkyne to form the trans -vinyl species which, upon further protonation, gives the corresponding alkene.

The stoichiometry of binding of flavin mononucleotide (FMN) hydroquinone to Escherichia coli chorismate synthase

Abstract Escherichia coli chorismate synthase (EC 4.6.1.4), purified aerobically, does not contain oxidised flavin mononucleotide (FMN) as judged by its uv/visible and fluorescence spectra. However, transient kinetic studies of functioning enzyme show that each enzyme tetramer binds four equivalents of FMNH2, the reduced hydroquinone state. The higher affinity of chorismate synthase for FMNH2 (KD 20 μM) is similar to that of bacterial luciferase.

Mechanistic studies on nitrogenase from Klebsiella pneumoniae using the rapid quench technique

ABSTRACT A mechanism for the nitrogenase-catalysed reduction of dinitrogen (N2) to ammonia is presented. The mechanism is based on pre-steady state kinetic data for dihydrogen (H2), hydrazine (N2H4) and ammonia (NH3) formation obtained by a rapid quench technique and on a consideration of the chemistry of transition metal dinitrogen, dinitrogen hydride and hydride complexes. Stopped-flow spectrophotometry has demonstrated that electron transfer between the component proteins of nitrogenase is coupled to the hydrolysis of MgATP and that the dissociation of the oxidised Fe protein from reduced Mo-Fe protein is the rate limiting step in the catalytic cycle. A hydrazido(2–) species is suggested as an intermediate in N2 reduction and a nitride or nitrene species as an intermediate in both N2 and azide reduction.

A precursor in the photolytic cleavage of the Co(III)C bond of coenzyme B-12 unobservable by electron paramagnetic resonance

Photolysis of a frozen (80--200 K) anaerobic solution of 5-deoxyadenosyl-cobalamin in aqueous propan-1,2-diol produces only a small Co(II) signal detectable by electron paramagnetic resonance (EPR). Upon warming to room temperature and refreezing without further irradiation the Co(II) signal increases many-fold. The interpretation is that at low temperature there is an EPR-undetectable incipient homolysis of the Co-C bond of the coenzyme which is revealed at higher temperature. The possible implications of this observation for the coenzyme B-12-dependent enzymes are noted.