Positive cooperativity during Azotobacter vinelandii nitrogenase-catalyzed acetylene reduction.

The MoFe protein component of the nitrogenase enzyme complex is the substrate reducing site and contains two sets of symmetrically arrayed metallo centers called the P (Fe8S7) and the FeMoco (MoFe7S9-C-homocitrate) centers. The ATP-binding Fe protein is the specific reductant for the MoFe protein. Both symmetrical halves of the MoFe protein are thought to function independently during nitrogenase catalysis. Forming [AlF4]- transition-state complexes between the MoFe protein and the Fe protein of Azotobacter vinelandii ranging from 0 to 2 Fe protein/MoFe protein produced a series of complexes whose specific activity decreases with increase in bound Fe protein/MoFe protein ratio. Reduction of 2H+ to H2 was inhibited in a linear manner with an x-intercept at 2.0 with increasing Fe protein binding, whereas acetylene reduction to ethylene decreased more rapidly with an x-intercept near 1.5. H+ reduction is a distinct process occurring independently at each half of the MoFe protein but acetylene reduction decreases more rapidly than H+ reduction with increasing Fe protein/MoFe protein ratio, suggesting that a response is transmitted between the two αß halves of the MoFe protein for acetylene reduction as Fe protein is bound. A mechanistic model is derived to investigate this behavior. The model predicts that each site functions independently for 2H+ reduction to H2. For acetylene reduction, the model predicts positive (synchronous) not negative cooperativity arising from acetylene binding to both sites before substrate reduction occurs. When this model is applied to inhibition by Cp2 and modified Av2 protein (L127∆) that form strong, non-dissociable complexes, positive cooperativity is absent and each site acts independently. The results suggest a new paradigm for the catalytic function of the MoFe protein during nitrogenase catalysis.

An epidemiological study of the magnitude and consequences of work related violence: the Minnesota Nurses' Study.

AIMS: To identify the magnitude of and potential risk factors for violence within a major occupational population. METHODS: Comprehensive surveys were sent to 6300 Minnesota licensed registered (RNs) and practical (LPNs) nurses to collect data on physical and non-physical violence for the prior 12 months. Re-weighting enabled adjustment for potential biases associated with non-response, accounting for unknown eligibility. RESULTS: From the 78% responding, combined with non-response rate information, respective adjusted rates per 100 persons per year (95% CI) for physical and non-physical violence were 13.2 (12.2 to 14.3) and 38.8 (37.4 to 40.4); assault rates were increased, respectively, for LPNs versus RNs (16.4 and 12.0) and males versus females (19.4 and 12.9). Perpetrators of physical and non-physical events were patients/clients (97% and 67%, respectively). Consequences appeared greater for non-physical than physical violence. Multivariate modelling identified increased rates for both physical and non-physical violence for working: in a nursing home/long term care facility; in intensive care, psychiatric/behavioural or emergency departments; and with geriatric patients. CONCLUSIONS: Results show that non-fatal physical assault and non-physical forms of violence, and relevant consequences, are frequent among both RNs and LPNs; such violence is mostly perpetrated by patients or clients; and certain environmental factors appear to affect the risk of violence. This serves as the basis for further analytical studies that can enable the development of appropriate prevention and control efforts.

Reduction of nitrogenase Fe protein from Azotobacter vinelandii by dithionite: quantitative and qualitative effects of nucleotides, temperature, pH and reaction buffer.

Oxidized Fe protein from Azotobacter vinelandii (Av2(0)) was reduced by dithionite (DT) in the absence and presence of nucleotides, over the temperature range 10-40 degrees C, over the pH range 7-8, and in various buffers--inorganic phosphate, TES, HEPES, and Tris. The reduction of each species of Fe protein--Av2(0), Av2(0)(MgATP)2, and Av2(0)(MgADP)2--was resolved into at least three exponential phases, with relative amplitudes of each phase varying over the range of experimental conditions, suggesting a dynamic population shift of kinetically distinct species. The rapid phase of Av2(0) reduction predominated at low temperature and pH, and in Tris buffer; rapid Av2(0)(MgATP)2 reduction was favored at high temperature and pH, and in phosphate buffer; and Av2(0)(MgADP)2 reduction was favored under more physiologically relevant conditions of 20 degrees C, pH 7.5, and in phosphate buffer. The rates of reduction of Fe protein species did not change with buffer, but temperature and pH do have an effect on the rates. With the appropriate constants, an empirically derived equation estimates the rate of Fe protein reduction at any temperature and pH within the limits 10-40 degrees C and pH 7-8, for a given species of Fe protein, and a given phase of the reaction. At 23.0 degrees C and pH 7.4, the rate of the dominant phase of Av2(0) reduction is 1.9 x 10(8) M(-1) s(-1). Under the same conditions, the rates of the two dominant phases of Av2(0)(MgATP)2 reduction are 1.2 x 10(6) and 1.5 x10 (5) M(-1) s(-1); and the rate of the dominant phase of Av2(0)(MgADP)2 reduction is 3.5 x 10(6) in M(-1) s(-1). Thermodynamic activation parameters for each phase of reduction were calculated. No breaks in the Arrhenius plots for any Fe protein species were observed.

Duplication and extension of the Thorneley and Lowe kinetic model for Klebsiella pneumoniae nitrogenase catalysis using a MATHEMATICA software platform.

The Thorneley and Lowe kinetic model for nitrogenase catalysis was developed in the early to mid 1980s, and has been of value in accounting for many aspects of nitrogenase catalysis. It has also been of value by providing a model for predicting new catalytic behavior. Since its original publication, new results have been obtained and have been successfully incorporated into the model. However, the computer program used for nitrogenase simulations has not been generally available. Using kinetic schemes and assumptions previously outlined by Thorneley and Lowe, we report attempts to duplicate the original T&L kinetic simulation for Klebsiella pneumoniae nitrogenase catalysis using an updated simulation based on the MATHEMATICA programming format, which makes it more user-friendly and more readily available. Comparisons of our simulations with the original T&L simulations are generally in agreement, but in some cases serious discrepancy is observed. Possible reasons for the differences are discussed. In addition to duplicating the original T&L model, we report effects of updating it by including information that has come to light subsequent to its original publication.

Hydrogen peroxide formation during iron deposition in horse spleen ferritin using O2 as an oxidant.

The reaction of Fe2+ with O2 in the presence of horse spleen ferritin (HoSF) results in deposition of FeOH3 into the hollow interior of HoSF. This reaction was examined at low Fe2+/HoSF ratios (5-100) under saturating air at pH 6.5-8.0 to determine if H2O2 is a product of the iron deposition reaction. Three methods specific for H2O2 detection were used to assess H2O2 formation: (1) a fluorometric method with emission at 590 nm, (2) an optical absorbance method based on the reaction H2O2 + 3I- + 2H+ = I3- + 2H2O monitored at 340 nm for I3- formation, and (3) a differential pulsed electrochemical method that measures O2 and H2O2 concentrations simultaneously. Detection limits of 0.25, 2.5, and 5.0 microM H2O2 were determined for the three methods, respectively. Under constant air-saturation conditions (20% O2) and for a 5-100 Fe2+/HoSF ratio, Fe2+ was oxidized and the resulting Fe3+ was deposited within HoSF but no H2O2 was detected as predicted by the reaction 2Fe2+ + O2 + 6H2O = 2Fe(OH)3 + H2O2 + 4H+. Two other sets of conditions were also examined: one with excess but nonsaturating O2 and another with limiting O2. No H2O2 was detected in either case. The absence of H2O2 formation under these same conditions was confirmed by microcoulometric measurements. Taken together, the results show that under low iron loading conditions (5-100 Fe2+/HoSF ratio), H2O2 is not produced during iron deposition into HoSF using O2 as an oxidant. This conclusion is inconsistent with previous, carefully conducted stoichiometric and kinetic measurements [Xu, B., and Chasteen, N. D. (1991) J. Biol. Chem. 266, 19965], predicting that H2O2 is a quantitative product of the iron deposition reaction with O2 as an oxidant, even though it was not directly detected. Possible explanations for these conflicting results are considered.

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.

Mechanistic interpretation of the dilution effect for Azotobacter vinelandii and Clostridium pasteurianum nitrogenase catalysis.

Nitrogenase activity for Clostridium pasteurianum (Cp) at a Cp2:Cp1 ratio of 1.0 and Azotobacter vinelandii (Av) at Av2:Av1 protein ratios (R) of 1, 4 and 10 is determined as a function of increasing MoFe protein concentration from 0.01 to 5 microM. The rates of ethylene and hydrogen evolution for these ratios and concentrations were measured to determine the effect of extreme dilution on nitrogenase activity. The experimental results show three distinct types of kinetic behavior: (1) a finite intercept along the concentration axis (approximately 0.05 microM MoFe); (2) a non-linear increase in the rate of product formation with increasing protein concentration (approximately 0.2 microM MoFe) and (3) a limiting linear rate of product formation at high protein concentrations (>0.4 microM MoFe). The data are fitted using the following rate equation derived from a mechanism for which two Fe proteins interact cooperatively with a single half of the MoFe protein. (see equation) The equation predicts that the cubic dependence in MoFe protein gives rise to the non-linear rate of product formation (the dilution effect) at very low MoFe protein concentrations. The equation also predicts that the rate will vary linearly at high MoFe protein concentrations with increasing MoFe protein concentration. That these limiting predictions are in accord with the experimental results suggests that either two Fe proteins interact cooperatively with a single half of the MoFe protein, or that the rate constants in the Thorneley and Lowe model are more dependent upon the redox state of MoFe protein than previously suspected [R.N. Thornley and D. J. Lowe, Biochem. J. 224 (1984) 887-894]. Previous Klebsiella pneumoniae and Azotobacter chroococcum dilution results were reanalyzed using the above equation. Results from all of these nitrogenases are consistent and suggest that cooperativity is a fundamental kinetic aspect of nitrogenase catalysis.

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.

Reactions of Azotobacter vinelandii nitrogenase using Ti(III) as reductant.

Nitrogenase-catalyzed reactions using Ti(III) were examined under a wide variety of conditions to determine the suitability of Ti(III) to serve as a general nitrogenase reductant. Solutions prepared from H2-reduced TiCl3, aluminum-reduced TiCl3, TiCl2, evaporated TiCl3 from an HCl, solution, and TiF3 were evaluated as reductants. Three general types of reactivity were observed. The first showed that, below Ti(III) concentrations of about 0.50 mM, nitrogenase catalysis utilized Ti(III) in a first-order reaction. The second showed that, above 0.50 mM, the rate of nitrogenase catalysis was zero order in Ti(III), indicating the enzyme was saturated with this reductant. Above 2.0-5.0 mM, nitrogenase catalysis was inhibited by Ti(III) depending on the titanium source used for solution preparation. This inhibition was investigated and found to be independent of the buffer type and pH, while high salt and citrate concentrations caused moderate inhibition. [Ti(IV)] above 2.0-3.0 mM and [Ti(III)] above about 5.0 mM were inhibitory. ATP/2e values were 4-5 for [Ti(III)] at or below 1.0-2.0 mM, 2.0 from 5.0 to 7.0 mM Ti(III) where nitrogenase is not inhibited, and 2.0 above 7.0 mM Ti(III) where severe inhibition occurs. For nitrogenase-catalyzed reactions using Ti(III) as reductant, the potential of the solution changes with time as the Ti(III)/Ti(IV) ratio changes. From the change in the rate of product formation (Ti(III) disappearance) with change in solution potential, the rate of nitrogenase catalysis was determined as a function of solution potential. From such experiments, a midpoint turnover potential of -480 mV was determined for nitrogenase catalysis with an associated n = 2 value.

Enhanced efficiency of ATP hydrolysis during nitrogenase catalysis utilizing reductants that form the all-ferrous redox state of the Fe protein.

The amount of MgATP hydrolyzed per pair of electrons transferred (ATP/2e) during nitrogenase catalysis (1.0 atm N(2), 30 degrees C) using titanium(III) citrate (Ti(III)) as reductant was measured and compared to the same reaction using dithionite (DT). ATP/2e values near 2.0 for Ti(III) and 5.0 for DT indicate that nitrogenase has a much lower ATP requirement using Ti(III) as reductant. Using reduced Azotobacter vinelandii flavoprotein (AvFlpH(2)), a possible in vivo nitrogenase reductant, ATP/2e values near 2.0 were also observed. When the reaction was conducted using Ti(III) under N(2), 5% CO in N(2), Ar, 5% CO in Ar, or acetylene, ATP/2e values near 2.0 were also observed. With Ti(III) as reductant, ATP/2e values near 2.0 were measured as a function of temperature, Fe:MoFe protein ratio, and MoFe:Fe protein ratio, in contrast to measured values of 4.0-25 when DT is used under the same conditions. Both Ti(III) and AvFlpH(2) are capable of forming the [Fe(4)S(4)](0) cluster state of the Fe protein whereas DT is not, suggesting that ATP/2e values near 2.0 arise from operation of the [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couple with hydrolysis of only 2 ATPs per pair of electrons transferred. Additional experiments showed that ATP/2e values near 2. 0 correlated with slower rates of product formation and that faster rates of product formation produced ATP/2e values near 5.0. ATP/2e values of 5.0 are consistent with the operation of the [Fe(4)S(4)](2+)/[Fe(4)S(4)](1+) redox couple while ATP/2e values of 2.0 could arise from operation of the [Fe(4)S(4)](2+)/[Fe(4)S(4)](0) redox couple. These results suggest that two distinct Fe protein redox couples may be functional during nitrogenase catalysis and that the efficiency of ATP utilization depends on which of these redox couples is dominant.

Forming the phosphate layer in reconstituted horse spleen ferritin and the role of phosphate in promoting core surface redox reactions.

Apo horse spleen ferritin (apo HoSF) was reconstituted to various core sizes (100-3500 Fe3+/HoSF) by depositing Fe(OH)3 within the hollow HoSF interior by air oxidation of Fe2+. Fe2+ and phosphate (Pi) were then added anaerobically at a 1:4 ratio, and both Fe2+ and Pi were incorporated into the HoSF cores. The resulting Pi layer consisted of Fe2+ and Pi at about a 1:3 ratio which is strongly attached to the reconstituted ferritin mineral core surface and is stable even after air oxidation of the bound Fe2+. The total amount of Fe2+ and Pi bound to the iron core surface increases as the core volume increases up to a maximum near 2500 iron atoms, above which the size of the Pi layer decreases with increasing core size. Mössbauer spectroscopic measurements of the Pi-reconstituted HoSF cores using 57Fe2+ show that 57Fe3+ is the major species present under anaerobic conditions. This result suggests that the incoming 57Fe2+ undergoes an internal redox reaction to form 57Fe3+ during the formation of the Pi layer. Addition of bipyridine removes the 57Fe3+ bound in the Pi layer as [57Fe(bipy)3]2+, showing that the bound 57Fe2+ has not undergone irreversible oxidation. This result is related to previous studies showing that 57Fe2+ bound to native core is reversibly oxidized under anaerobic conditions in native holo bacterial and HoSF ferritins. Attempts to bury the Pi layer of native or reconstituted HoSF by adding 1000 additional iron atoms were not successful, suggesting that after its formation, the Pi layer "floats" on the developing iron mineral core.

Redox reactivity of animal apoferritins and apoheteropolymers assembled from recombinant heavy and light human chain ferritins.

The redox reactivities of air-oxidized apo horse spleen ferritin (HoSF) and apo rat liver ferritin (RaF) were examined by microcoulometry and reductive optical titrations. Microcoulometry on several independent lots of commercial HoSF revealed two distinct types of redox activity: one requiring 3-4 electrons and one requiring 6-7 electrons for full reduction of the protein shell. ApoRaF required 8-9 electrons to fully reduce the oxidized form. Reductive optical titrations confirmed the microcoulometric reduction stoichiometry and, in addition, showed that the spectra of both oxidized and reduced apoHoSF were distinct and possessed absorbances tailing into the visible region. The redox reactivity of both apoRaF and apoHoSF correlated with their H-subunit composition. Identical microcoulometric and optical experiments were conducted with recombinant apo human liver heavy (rHuHF) and light (rHuLF) ferritins, but neither was redox-active. These results suggest that the redox reactivity of native ferritins is due to their heteropolymeric nature. This was confirmed by mixing various proportions of rHuHF and rHuLF, dissociating the 24-mers into individual subunits with guanidine hydrochloride at pH 3.5, and renaturing to form heteropolymeric 24-mers. Microcoulometric measurements of these apoheteropolymers reassembled in vitro showed that they were redox-active like their native apoheteropolymer counterparts. The redox activity of these apoheteropolymers increased with H-subunit composition, reached a maximum near 12 H- and 12 L-subunits, and then declined to zero with increasing L-subunit composition. The decline in redox reactivity at high L-subunit concentrations indicates that both H- and L-subunits are involved in forming the observed redox centers. Apoheteropolymers formed from rHuLF and W93F (an H-chain mutant) were redox-inactive, suggesting that the conserved tryptophan is necessary for redox center formation.

Steady-state kinetic studies of dithionite utilization, component protein interaction, and the formation of an oxidized iron protein intermediate during Azotobacter vinelandii nitrogenase catalysis.

Steady-state kinetic analysis of the two-component protein system of Azotobacter vinelandii (Av) nitrogenase is reported. A precisely obeyed half-order reaction in dithionite was observed at concentrations up to 21 mM with no indication of saturation by this substrate. This behavior was monitored by optical, amperometric, and manometric kinetic techniques, and the results were mathematically fit to establish the half-order reaction in dithionite. Under conditions where the MgATP and dithionite concentrations remain unchanged, Av2 (the Fe protein component) interacts with Av1 (the MoFe protein component according to the rate law, suggesting a rapid 1:1 Av2-Av1 interaction: [formula: see text]. with [Av2] the free Fe protein concentration, K = 5.9 microM, and Vmax = 2314 nmol of H2 min-1 (mg of Av1)-1. Under dithionite-depleted conditions, Av2 undergoes an Av1-mediated, one-electron oxidation, consistent with its proposed role as a specific, single-electron reductant for Av1. During steady-state turnover as a function of Av2/Av1 ratio, optical spectroscopy demonstrated the presence of 25-30% oxidized Av2 as an enzyme intermediate. Computer-averaged EPR spectra showed that Av1 was > 95% EPR-silent and Av2 was up to 30% oxidized (Av2ox), consistent with the optical measurements. These optical and EPR results show that up to six Av2ox per Av1 can accumulate in the presence of dithionite during catalysis, suggesting that the conversion of Av2ox back into Av2red is a relatively slow process.

A kinetic study of iron release from Azotobacter vinelandii bacterial ferritin.

The kinetics of iron release from Azotobacter vinelandii bacterial ferritin (AVBF) was measured by reduction of core iron with S2O4(2-) followed by chelation of Fe2+ with alpha, alpha-bipyridine (bipy). The rate was first order in AVBF and one half order in S2O4(2-), suggesting that SO2- is the active reductant formed by S2O4(2-) = 2SO2-. With zero-order conditions for dithionite and bipy, two consecutive first-order iron release reactions differing by a factor of about 14 were observed with rate constants of 0.0263 and 0.00184 sec-1, respectively, at 25 degrees C and pH 7.0. The faster reaction corresponded to the loss of 1433 iron atoms (91%) and the slower second reaction corresponded to loss of 145 (9%) of the original 1575 iron atoms present. The first reaction increased about twofold with pH variation between 6.5 and 8.0, whereas the second reaction was unchanged in the pH range 5.5-8. Both dramatically increased at pH 5.0. Methyl viologen increased the rate of both reactions about tenfold. The biphasic behavior for iron loss is interpreted as two different populations of iron atoms present in the core of AVBF, the first representing the bulk iron, and the second a group of unique iron atoms released last which may represent iron attached to the interior of the protein shell or iron associated with the heme groups. Kinetic stopped-flow measurements show that the heme is first reduced, followed by reduction of the core iron by reduced heme, suggesting an electron transfer role for heme in AVBF function.

The concentration of cellular nitrogenase proteins in Azotobacter vinelandii whole cells as determined by activity measurements and electron paramagnetic resonance spectroscopy.

The concentration of MoFe protein (Av1) in Azotobacter vinelandii whole-cell crude extract was measured by electron paramagnetic resonance spectroscopy at g = 3.7 resonance. The Av1 concentration was also measured from the activity of crude extract to which increasing amounts of purified Av1 and Av2 were added. The Av2 concentration was determined by fitting activity measurements of crude extract and crude extract to which purified Av2 was added. The Av1 concentration was found to be 26-28 microM and that for Av2 was 42-45 microM in whole cells, with a Av2/Av1 ratio of 1.6. In vitro activity measurements carried out as a function of Av1 concentration at Av2/Av1 ratios of 1 and 4 showed a dilution effect below 0.08 microM, a factor of 2 below that observed for nitrogenase reactivity for Klebsiella pneumoniae. No deviations from linearity were observed up to 26 microM for the Av1-Av2 interaction. The flavoprotein (AvFlp) was shown to enhance nitrogenase reactivity at low Av2/Av1 ratios, a result attributed to decreasing the Km for Av2-Av1 interaction. Direct reduction of bound Av2 is possibly the source of this kinetic enhancement. The kinetic results are considered in terms of the Thorneley and Lowe scheme.

Reductant-independent ATP hydrolysis catalyzed by homologous nitrogenase proteins from Azotobacter vinelandii and heterologous crosses with Clostridium pasteuranium.

Reductant-independent ATPase activity was initiated and studied for Azotobacter vinelandii and Clostridium pasteuranium nitrogenase proteins (Av1, Cp1 and Av2, Cp2, 1 designating the iron molybdenum protein and 2 the iron protein) and their heterologous crosses by two methods: (1) allowing dithionite to be depleted from a normal assay in the presence of substrate levels of MgATP and (2) using reduced but reductant-free nitrogenase proteins in the presence of substrate levels of MgATP. In both cases, at a 1:1 protein ratio, MgATP is converted initially to MgADP with a specific activity of 400-500 nmol MgATP hydrolyzed/min.mg Av1, but in slower steps the MgADP is converted to AMP and, after 12 h, AMP is ultimately converted to adenosine. This reactivity requires the presence of both proteins, increases with increasing Av2/Av1 ratio, and is not a result of unique redox states of either protein. For Av1-Av2, ATP hydrolysis in the absence of Mg2+ occurred at nearly the same rate as reductant-dependent MgATP hydrolysis. Reductant-independent ATPase activity also occurred for the Av1-Cp2 and Cp1-Av2 heterologous crosses and was 2-fold and 18-fold slower than the Av1-Av2 or Cp1-Cp2 combinations. In both cases further hydrolysis of MgADP to AMP and AMP to adenosine occurred. A unique nucleotide hydrolysis system is apparently operating in the complex formed between the two nitrogenase proteins in the absence of reductant. The relationship between the reductant-independent and reductant-dependent activities of nitrogenase catalysis is explored.

Metal ion binding to apo, holo, and reconstituted horse spleen ferritin.

The binding of Cd2+, Zn2+, Cu2+, Ni2+, Co2+, Mn2+, and Mg2+ to apo, holo, reconstituted horse spleen ferritin (HoSF), and native holo HoSF with phosphate removed was measured by gel-exclusion chromatography. Three classes of strong binding interactions (Kd < 10(-7) M) with apo HoSF at pH 7.5 were found for the various M2+ studied: high stoichiometric binding (30-54 M2+/HoSF) for Cd2+, Zn2+, Cu2+, with two protons released per metal bound; intermediate binding (16 M2+/HoSF) for Ni2+ and Co2+, with one proton released per metal bound; and low levels of binding (2-12 M2+/HoSF) for Mn2+, Mg2+, and Fe2+, with < 0.5 protons released per metal bound. M2+ binding to apo HoSF was nearly abolished at pH 5.5, except for Fe2+ and Cu2+, which remained unaffected by pH alteration. Holo HoSF bound much higher levels of M2+, a result directly attributable to the presence of phosphate binding sites. This conclusion was confirmed by decreased binding of M2+ to HoSF reconstituted in the absence of phosphate and by native holo HoSF with phosphate chemically removed. The binding of Cd2+ to apo HoSF was 54 per HoSF, but in the presence of developing core, the amount bound decreased to about 30 Cd2+/HoSF. This result indicated that Cd2+ and developing core were competing for the same sites on the HoSF interior, suggesting that 24 of the Cd2+ were bound to the inside surface. No other M2+ studied bound to the interior of HoSF by this criterion. Several of the M2+ appeared to bind strongly to the phosphate-free mineral core surface in reconstituted HoSF.

Evidence for a central role of lysine 15 of Azotobacter vinelandii nitrogenase iron protein in nucleotide binding and protein conformational changes.

Biological nitrogen fixation catalyzed by purified nitrogenase requires the hydrolysis of a minimum of 16 MgATP for each N2 reduced. In the present study, we demonstrate a central function for Lys-15 of Azotobacter vinelandii nitrogenase iron protein (FeP) in the interaction of nucleotides with nitrogenase. Changing Lys-15 of the FeP to Arg resulted in an FeP with a dramatically reduced affinity for both MgATP and MgADP. From equilibrium column binding experiments at different nucleotide concentrations, apparent dissociation constants (Kd) for wild type FeP binding of MgADP (143 microM) and MgATP (571 microM) were determined. Over the same nucleotide concentration ranges, the K15R FeP showed no significant affinity for either nucleotide. This contrasts sharply with previous results with an FeP in which Lys-15 was changed to Gln (K15Q) where it was found that the K15Q FeP bound MgADP with the same affinity as wild type FeP and MgATP with a slightly reduced affinity. Analysis of K15R FeP by EPR, circular dichroism (CD), and microcoulometry revealed that the [4Fe-4S] cluster was unaffected by the amino acid change and that addition of either MgADP or MgATP did not result in the protein conformational changes normally detected by these techniques. These results are integrated into a model for how MgATP and MgADP bind and induce conformational changes within the FeP.

Iron core formation in horse spleen ferritin: magnetic susceptibility, pH, and compositional studies.

Horse spleen ferritin (HoSF) reconstituted with small iron cores ranging in size from 8 to 500 iron atoms was studied by magnetic susceptibility and pH measurements to determine when the added Fe3+ begins to aggregate and form antiferromagnetically coupled clusters and also to determine the hydrolytic state of the iron at low iron loading. The Evans NMR magnetic susceptibility measurements showed that at iron loadings as low as 8 Fe3+/HoSF, at least half of the added iron atoms were involved in antiferromagnetic exchange interactions and the other half were present as isolated iron atoms with S = 5/2. As the core size increased to about 24 iron atoms, the antiferromagnetic exchange interactions among the iron atoms increased until reaching the limiting value of 3.8 Bohr magnetons per iron atom, the value present in holo HoSF. HoSF containing eight or more Fe3+ to which eight Fe2+ were added showed that the Fe2+ ions were at sites remote from the Fe3+ and that the resulting HoSF consisted of individual, noninteracting Fe2+ and the partially aggregated Fe3+. pH measurements for core reduction showed that Fe(OH)3 was initially present at all iron loadings but that in the absence of iron chelators the reduced iron core is partially hydrolyzed. Proton induced x-ray emission spectroscopy showed that Cl- is transported into the iron core during reduction, forming a stable chlorohydroxy Fe(II) mineral phase.

A capillary electrophoresis method for studying apo, holo, recombinant, and subunit dissociated ferritins.

A capillary electrophoresis (CE) method is described for detecting and quantitating apo and holo ferritins from horse spleen (HoSF), rat liver (RLF), recombinant human light chain (rLF), recombinant human heavy chain (rHF), site-directed variants of human light chain, and Azotobacter vinelandii bacterial ferritin (AVBF). This procedure is carried out at pH 8.2, where the ferritin molecules are associated into their 24-mers. Protein mobilities as expressed as elution times were clearly resolved and could be used to distinguish one ferritin type from another, providing a means for detecting and quantitating various ferritin species in purified or partially purified states. Measurements of these and other ferritins were also conducted at pH 2.0, where dissociation into their respective subunits occurs. For HoSF and RLF, the individual L and H subunits were resolved and their relative concentrations were determined by integrating the areas of the elution peaks. HoSF gave 89.8% L and 10.2% H and RLF gave 70.7% L and 29.3% H, while rLF, rHF, and AVBF gave only a single subunit, all in agreement with reported values obtained by polyacrylamide gel electrophoresis. CE of HoSF, containing increasing amounts of iron in the interior, in general, showed that protein mobilities increased, reached a plateau, and then slowly decreased with increasing core size, although buffer effects altered this CE behavior to some extent. Such results indicate that species formed early during core formation have individual iron atoms present and differ from those formed later in which the oligomeric iron core has formed.(ABSTRACT TRUNCATED AT 250 WORDS)