Electron transfer and half-reactivity in nitrogenase

Nitrogenase is a globally important enzyme that catalyses the reduction of atmospheric dinitrogen into ammonia and is thus an important part of the nitrogen cycle. The nitrogenase enzyme is composed of a catalytic molybdenum-iron protein (MoFe protein) and a protein containing an [Fe4-S4] cluster (Fe protein) that functions as a dedicated ATP-dependent reductase. The current understanding of electron transfer between these two proteins is based on stopped-flow spectrophotometry, which has allowed the rates of complex formation and electron transfer to be accurately determined. Surprisingly, a total of four Fe protein molecules are required to saturate one MoFe protein molecule, despite there being only two well-characterized Fe-protein-binding sites. This has led to the conclusion that the purified Fe protein is only half-active with respect to electron transfer to the MoFe protein. Studies on the electron transfer between both proteins using rapid-quench EPR confirmed that, during pre-steady-state electron transfer, the Fe protein only becomes half-oxidized. However, stopped-flow spectrophotometry on MoFe protein that had only one active site occupied was saturated by approximately three Fe protein equivalents. These results imply that the Fe protein has a second interaction during the initial stages of mixing that is not involved in electron transfer.     

Electron transfer within nitrogenase: evidence for a deficit-spending mechanism

The reduction of substrates catalyzed by nitrogenase utilizes an electron transfer (ET) chain comprised of three metalloclusters distributed between the two component proteins, designated as the Fe protein and the MoFe protein. The flow of electrons through these three metalloclusters involves ET from the [4Fe-4S] cluster located within the Fe protein to an [8Fe-7S] cluster, called the P cluster, located within the MoFe protein and ET from the P cluster to the active site [7Fe-9S-X-Mo-homocitrate] cluster called FeMo-cofactor, also located within the MoFe protein. The order of these two electron transfer events, the relevant oxidation states of the P-cluster, and the role(s) of ATP, which is obligatory for ET, remain unknown. In the present work, the electron transfer process was examined by stopped-flow spectrophotometry using the wild-type MoFe protein and two variant MoFe proteins, one having the β-188(Ser) residue substituted by cysteine and the other having the β-153(Cys) residue deleted. The data support a "deficit-spending" model of electron transfer where the first event (rate constant 168 s(-1)) is ET from the P cluster to FeMo-cofactor and the second, "backfill", event is fast ET (rate constant >1700 s(-1)) from the Fe protein [4Fe-4S] cluster to the oxidized P cluster. Changes in osmotic pressure reveal that the first electron transfer is conformationally gated, whereas the second is not. The data for the β-153(Cys) deletion MoFe protein variant provide an argument against an alternative two-step "hopping" ET model that reverses the two ET steps, with the Fe protein first transferring an electron to the P cluster, which in turn transfers an electron to FeMo-cofactor. The roles for ATP binding and hydrolysis in controlling the ET reactions were examined using βγ-methylene-ATP as a prehydrolysis ATP analogue and ADP + AlF(4)(-) as a posthydrolysis analogue (a mimic of ADP + P(i)).     

Evidence for a two-electron transfer using the all-ferrous Fe protein during nitrogenase catalysis

The nitrogenase-catalyzed H(2) evolution and acetylene-reduction reactions using Ti(III) and dithionite (DT) as reductants were examined and compared under a variety of conditions. Ti(III) is known to make the all-ferrous Fe protein ([Fe(4)S(4)](0)) and lowers the amount of ATP hydrolyzed during nitrogenase catalysis by approximately 2-fold. Here we further investigate this behavior and present results consistent with the Fe protein in the [Fe(4)S(4)](0) redox state transferring two electrons ([Fe(4)S(4)](2+)/[Fe(4)S(4)](0)) per MoFe protein interaction using Ti(III) but transferring only one electron ([Fe(4)S(4)](2+)/[Fe(4)S(4)](1+)) using DT. MoFe protein specific activity was measured as a function of Fe:MoFe protein ratio for both a one- and a two-electron transfer reaction, and nearly identical curves were obtained. However, Fe protein specific activity curves as a function of MoFe:Fe protein ratio showed two distinct reactivity patterns. With DT as reductant, typical MoFe inhibition curves were obtained for operation of the [Fe(4)S(4)](2+)/[Fe(4)S(4)](1+) redox couple, but with Ti(III) as reductant the [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couple was functional and MoFe inhibition was not observed at high MoFe:Fe protein ratios. With Ti(III) as reductant, nitrogenase catalysis produced hyperbolic curves, yielding a V(max) for the Fe protein specific activity of about 3200 nmol of H(2) min(-1) mg(-1) Fe protein, significantly higher than for reactions conducted with DT as reductant. Lag phase experiments (Hageman, R. V., and Burris, R. H. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 2699-2702) were carried out at MoFe:Fe protein ratios of 100 and 300 using both DT and Ti(III). A lag phase was observed for DT but, with Ti(III) product formation, began immediately and remained linear for over 30 min. Activity measurements using Av-Cp heterologous crosses were examined using both DT and Ti(III) as reductants to compare the reactivity of the [Fe(4)S(4)](2+)/[Fe(4)S(4)](1+) and [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couples and both were inactive. The results are discussed in terms of the Fe protein transferring two electrons per MoFe protein encounter using the [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couple with Ti(III) as reductant.     

Electron paramagnetic resonance studies on nitrogenase. III. Function of magnesium adenosine 5′-triphosphate and adenosine 5′-diphosphate in catalysis by nitrogenase

The electron paramagnetic resonance spectra of azoferredoxin and molybdoferredoxin, components of the nitrogenase of Clostridium pasteurianum, disappear when the proteins are oxidized by certain dyes. When molybdoferredoxin and azoferredoxin were mixed in a 1 to 2 molar ratio, the electron paramagnetic resonance spectrum of the mixture was the sum of the two spectra with the exception of a slight change in the azoferredoxin signal. Addition of magnesium ATP and dithionite to this reconstituted nitrogenase resulted in a rapid change in the spectrum of both nitrogenase components; the molybdoferredoxin spectrum at all g-values decreased with a half-life less than 70 ms to 40% of its original size whereas the azoferredoxin signal changed in shape and size with a half-life of less than 40 ms. If an ATP-generating system was added instead of MgATP so that no ADP accumulated, then the molybdoferredoxin signal almost completely disappeared and the azoferredoxin signal changed in shape and slightly in size. These changes occurred at molar ratios of molybdoferredoxin to azoferredoxin from 1:14 to 1:0.2. If the reaction was allowed to consume the reductant, then the molybdoferredoxin signal(s) was restored but the azoferredoxin signal disappeared. The signal of azoferredoxin was restored and the signal of molybdoferredoxin again disappeared on addition of more reductant. The data suggest that for nitrogenase to catalyze the reduction of substrates, the magnesium ATP-reduced azoferredoxin complex is formed first and this complex then reacts with molybdoferredoxin to allow electron flow. In addition the data suggests that the rate-limiting reaction is an ATP-mediated electron flow from azoferredoxin to molybdoferredoxin. Finally the results show that no flow of electrons from azoferredoxin or molybdoferredoxin occurs when a mixture of ADP and ATP in a molar ratio of 2:1 is added initially or is reached by conversion of ATP to ADP and inorganic phosphate during reduction of protons. A mechanism consistent with these findings is proposed.     

Electron donation to nitrogenase in heterocysts of cyanobacteria

Various electron donors were found to stimulate C₂H₂ reduction (N₂ fixation) by isolated heterocysts from Anabaena variabilis and Anabaena cylindrica. Intermediates of glycolysis and the tricarboxylic acid cycle as well as unphosphorylated sugars like glucose, fructose and erythrose were among these electron donors. The transfer of electrons from donors like H₂, NADH, glyoxylate and glycollate was strictly light-dependent, whereas others like NADPH or pyruvate plus coenzyme A supported C₂H₂ reduction also in the dark. In all cases, the overall activity was enhanced by light. The stimulation by light was more distinct with heterocysts from A. variabilis than with heterocysts from A. cylindrica.The present communication establishes that pyruvate supports C₂H₂ reduction by heterocysts from either A. variabilis or A. cylindrica with rates comparable to those with other electron donors. Pyruvate could, however, support C₂H₂ reduction only in the presence of coenzyme A, and the concentrations of both coenzyme A and pyruvate were crucial. A pyruvate-dependent reduction of ferredoxin by extracts from heterocysts was recorded spectrophotometrically. Glyoxylate, which is an inhibitor of thiamine pyrophosphate-dependent decarboxylations, inhibited pyruvate-dependent C₂H₂ reduction. This result supports the conclusion that pyruvate is metabolised by pyruvate: ferredoxin oxidoreductase in heterocysts. High concentrations of pyruvate and other electron donors inhibited C₂H₂ reduction which suggests that nitrogenase activity in heterocysts may be controlled by the availability of electron donors.Dedicated to Professor Norbert Pfennig, Konstanz, on the occasion of his 60th birthday     

Electron microscopy of the Mo-Fe-protein from Azotobacter vinelandii nitrogenase

The quaternary structure of the Mo-Fe-protein from Azotobacter vinelandii has been studied by electron microscopy. A model of the molecule of the Mo-Fe-protein has been proposed: two alpha subunits are displaced relative to two beta subunits along a twofold axis, so the molecule can be characterized by the point-group pseudosymmetry 222. Computer averaging of the images showed that one of the projections of the molecule could be characterized by twofold rotational symmetry. Micrographs of nitrogenase recombined complex (Mo-Fe-protein + Fe-protein) have been obtained. They showed particles close in size and form to the Mo-Fe-protein molecule. Therefore, it has been proposed that the Fe-protein could be situated in the central cavity of Mo-Fe-protein.     

Electron transfer in quinoproteins

Soluble quinoprotein dehydrogenases oxidize a wide range of sugar, alcohol, amine, and aldehyde substrates. The physiological electron acceptors for these enzymes are not pyridine nucleotides but are other soluble redox proteins. This makes these enzymes and their electron acceptors excellent systems with which to study mechanisms of long-range interprotein electron transfer reactions. The tryptophan tryptophylquinone (TTQ)-dependent methylamine dehydrogenase (MADH) transfers electrons to a blue copper protein, amicyanin. It has been possible to alter the rate of electron transfer by using different redox forms of MADH, varying reaction conditions, and performing site-directed mutagenesis on these proteins. From kinetic and thermodynamic analyses of the reaction rates, it was possible to determine whether a change in rate is due a change in Delta G(0), electronic coupling, reorganization energy or kinetic mechanism. Examples of each of these cases are discussed in the context of the known crystal structures of the electron transfer protein complexes. The pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase transfers electrons to a c-type cytochrome. Kinetic and thermodynamic analyses of this reaction indicated that this electron transfer reaction was conformationally coupled. Quinohemoproteins possess a quinone cofactor as well as one or more c-type hemes within the same protein. The structures of a PQQ-dependent quinohemoprotein alcohol dehydrogenase and a TTQ-dependent quinohemoprotein amine dehydrogenase are described with respect to their roles in intramolecular and intermolecular protein electron transfer reactions.     

Electron transfer in DNA

Electrons migrate over long distances along the DNA in a multistep hopping process where the rate of each step depends strongly upon its length. The efficiency of this process is not only determined by the electron transfer rates but also by competing reactions with water, in which the charge carriers are trapped. Because electron transfer through DNA can occur under the conditions of oxidative stress, biological consequences are highly likely. In addition, it has been observed that some DNA-binding enzymes influence this charge transport. The question of whether DNA is a suitable material for nanolelectronic devices remains unanswered.     

Electron transfer in biology

Table VII presents a list of the topics I have discussed. Underlying biological electron transfer which employs metal ions overwhelmingly is the intimacy of the interaction between metal ion properties and protein properties. Attacking the problems is attacking a cornerstone of life - bioenergetics. It is appropriate that this is the Heyrovsky Memorial Lecture since he devised the polarograph which is a device for coupling electrolytes (protons) in solution with electrons in metal atoms.     

Analysis of steady state Fe and MoFe protein interactions during nitrogenase catalysis

Steady state kinetic measurements are reported for nitrogenase from Azotobacter vinelandii (Av) and Clostridium pasteurianum (Cp) under a variety of conditions, using dithionite as reductant. The specific activities of Av1 and Cp1 are determined as functions of Av2:Av1 and Cp2:Cp1, respectively, at component protein ratios from 0.4 to 50, and conform to a simple hyperbolic rate law for the interaction of Av2 with Av1 and Cp2 with Cp1. The specific activities of Av2 and Cp2 are also measured as a function of increasing Av1 and Cp1 concentrations, producing 'MoFe inhibition' at large MoFe:Fe ratios. When the rate of product formation under MoFe inhibited conditions is re-plotted as increasing Av2:Av1 or Cp2:Cp1 ratios, sigmoidal kinetic behavior is observed, suggesting that the rate constants in the Thorneley and Lowe (T&L) model are more dependent upon the oxidation level of MoFe protein than previously suspected [R.N.F. Thorneley, D.J. Lowe, Biochem. J. 224 (1984) 887-894], at least when applied to Av and Cp. Calculation of Hill coefficients gave values of 1.7-1.9, suggesting a highly cooperative Fe-MoFe protein interaction in both Av and Cp nitrogenase catalysis. The T&L model lacks analytical solutions [R.N.F. Thorneley, D.J. Lowe, Biochem. J. 215 (1983) 393-404], so the ease of its application to experimental data is limited. To facilitate the study of steady state kinetic data for H(2) evolution, analytical equations are derived from a different mechanism for nitrogenase activity, similar to that of Bergersen and Turner [Biochem. J. 131 (1973) 61-75]. This alternative cooperative model assumes that two Fe proteins interact with one MoFe protein active site. The derived rate laws for this mechanism were fitted to the observed sigmoidal behavior for low Fe:MoFe ratios (<0.4), as well as to the commonly observed hyperbolic behavior for high values of Fe:MoFe for both Av and Cp.     

Substrate interaction at an iron-sulfur face of the FeMo-cofactor during nitrogenase catalysis

Nitrogenase catalyzes biological dinitrogen fixation, the reduction of N(2) to 2NH(3). Recently, the binding site for a non-physiological alkyne substrate (propargyl alcohol, HC triple bond C-CH(2)OH) was localized to a specific Fe-S face of the FeMo-cofactor approached by the MoFe protein amino acid alpha-70(Val). Here we provide evidence to indicate that the smaller alkyne substrate acetylene (HC triple bond CH), the physiological substrate dinitrogen, and its semi-reduced form hydrazine (H(2)N-NH(2)) interact with the same Fe-S face of the FeMo-cofactor. Hydrazine is a relatively poor substrate for the wild-type (alpha-70(Val)) MoFe protein. Substitution of the alpha-70(Val) residue by an amino acid having a smaller side chain (alanine) dramatically enhanced hydrazine reduction activity. Conversely, substitution of alpha-70(Val) by an amino acid having a larger side chain (isoleucine) significantly lowered the capacity of the MoFe protein to reduce dinitrogen, hydrazine, or acetylene.     

Electron transfer mechanisms in electron transfer chains

Mechanisms responsible for the transfer of electrons through mitochondrial and photosynthetic electron transport chains are considered. Mechanisms considered include diffusion, ligand-mediated transfer, tunneling and semiconduction. Perturbations which create satisfactory conditions for electron transfer are also considered. There is a brief discussion of the electron transport chain environment and constituents. Sponsored in part by a grant from the Department of Health, Education, and Welfare (Public Health Service Grant Number 5 R01 RL00480)     

Electron transfer to nitrogenase in Klebsiella pneumoniae. nifF gene cloned and the gene product, a flavodoxin, purified.

The nifF gene of Klebsiella pneumoniae was cloned into a multicopy plasmid in order to construct a strain that synthesizes and retains an elevated concentration of the gene product relative to the wild-type strain. Characterization of the isolated flavodoxin, which serves as an electron donor to nitrogenase, shows unambiguously that it is the product of the nifF gene.     

Proton transfer in catalysis by fumarase.

Using 3T[14C]malate it was possible to show intermolecular T-transfer to unlabeled fumarate. The rate of dissociation of ET derived from the malate was not rapid, only about as fast as required for KMcat. Because of the slow dissociation of ET derived from T-malate, the awkward complex ET-malate is readily formed. As shown by the effect of added malate on the partition of ET, otherwise captured by fumarate, ET.malate must be functional. Its rate of dissociation to E.M determines the V/Km value of malate. Hydrogen dissociation of the complex ET.F was linearly related to the concentration and basicity of the buffer provided, varying from < 10% to > 60% of the overall rate with alkyl phosphonates. Partition of EH.F to free malate or fumarate occurs in a ratio approximately 2:1 at both low and high buffer. This agrees well with the comparison of the equilibrium exchange rates: malate with [18O]water to malate with [14C]-fumarate [Hansen, J.N., Dinovo, E.C., & Boyer, P.D. (1969) J. Biol. Chem. 244, 6270-6279]. Therefore, the abstracted hydroxyl group is fully exchanged from the enzyme when the bound hydrogen and fumarate return to malate and must be much more accessible to the medium than the abstracted proton. The fact that buffer increases the rate of proton transfer to the medium in the central complex makes it appear that a proton relay connects the active site donor with a remote site that interfaces with the ultimate proton source, water.     

Electron transfer reactions in methanogens

Abstract Methanogenic bacteria comprise a specialized group of obligately anaerobic microorganisms able to reduce a limited number of substrates to CH₄. The intermediates involved in this reduction process remain bound to a series of typical C₁-carriers. Reducing equivalents are either obtained from the oxidation of H₂ or from oxidation of carbon substrates to CO₂. Electron transfer reactions thus constitute the very essence of the process of methanogenesis.
In recent years much progress has been made in the elucidation of the special metabolic pathways and the nature of the C₁-carriers involved in methanogenic bacteria. The energy generated at the oxidoreduction reactions, notably at the methylreductase step, is conserved by ATP synthesis. The energy is used for cell carbon synthesis and, in catalytic amounts, for the reductive activation of some methanogenic enzymes. Before the condensing reaction resulting in the formation of acetyl-CoA takes place, 2 C₁-units are reduced or oxidized depending on the substrate to a carbonyl and a -CH₃ group. Formation of the latter proceeds via the methanogenic route. Intermediary cell carbon synthesis starting from acetyl-CoA involves reductive carboxylations and oxidoreductions by the participation of the enzymes of the tricarboxylic acid cycle.     

Electron transfer in ruthenium-modified proteins

Photochemical techniques have been used to measure the kinetics of intramolecular electron transfer in Ru(bpy)₂(im)(His)²⁺-modified (bpy = 2,2-bipyridine; im = imidazole) cytochromec and azurin. A driving-force study with the His33 derivatives of cytochromec indicates that the reorganization energy () for Fe²⁺Ru³⁺ ET reactions is 0.8 eV. Reductions of the ferriheme by either an excited complex,^*Ru²⁺, or a reduced complex, Ru⁺, are anomalously fast and may involve formation of an electronically excited ferroheme. The distance dependence of Fe²⁺Ru³⁺ and Cu⁺Ru³⁺ electron transfer in 12 different Ru-modified cytochromes and azurins has been analyzed using a tunneling-pathway model. The ET rates in 10 of the 12 systems exhibit an exponential dependence on metal-metal separation (decay constant of 1.06 å^–1) that is consistent with predictions of the pathway model.     

Electron transfer in photosystem II

The picture presently emerging from studies on the mechanism of photosystem II electron transport is discussed. The reactions involved in excitation trapping, charge separation and stabilization of the charge pair in the reaction center, followed by the reactions with the substrates, plastoquinone reduction and water oxidation, are described successively. Finally, a brief discussion on photosystem II heterogeneity is presented.