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.

Isolation and characterization of two rubredoxins from Clostridium thermoaceticum.

Two rubredoxins with similar molecular weights (about 6000) have been purified from Clostridium thermoaceticum, a thermophile and strict anaerobe. They exhibit minor differences in several properties like elution pattern from DEAE-cellulose column, isoelectric point, amino acid composition, absorption and EPR spectra and redox potential. Their chemical and physical properties are similar to those of other rubredoxins from anaerobic microorganisms.

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.

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.

Purification, composition, charge, and molecular weight of the FeMo cofactor from Azotobacter vinelandii nitrogenase.

A procedure has been developed for purifying NMF and NMF/DMF solutions of the FeMo cofactor (FeMoco) derived from the molybdenum iron protein of nitrogenase. This procedure consists of anaerobic chromatography of FeMoco solutions on two consecutive anaerobic molecular sizing columns followed by electrophoretic migration through a third sizing column. FeMoco prepared by this procedure is homogeneous as evidenced by chromatographic, electrophoretic, and compositional criteria. The minimal elemental composition was found to be MoFe6S6 using chemical colorimetric, inductively coupled plasma (ICP), and proton induced x-ray emission (PIXE) analytical procedures. Molecular weight measurements of NMF and DMF solutions of FeMoco using calibrated columns containing various molecular sizing matrices gave values of 1395 +/- 130 daltons for the molecular weight of FeMoco. The measured MW of FeMoco is about twice the value expected from the minimal stoichiometry, suggesting that FeMoco may exist as Mo2Fe12S12 in NMF and DMF solutions. The charge of FeMoco in its EPR silent state was determined to be 2- per Mo by passing NMF solutions of FeMoco containing excess salts of Na+, K+, Rb+, and Mg2+ through long columns equilibrated with pure NMF and then measuring the M/Mo ratio of the emerging FeMoco. Decomposition of purified FeMoco by acid or O2-exposure followed by exhaustive methylation or silanation of the resulting mixture failed to yield any methylated or silanated homocitric acid as measured by tandem gas chromatography-mass spectrometry (GC-MS) analysis. The GC-MS procedure applied to standard homocitric acid samples and various controls readily detects methylated homocitric acid at the sub-nanomole level. We conclude that the minimum molecular formula for active oxidized (EPR silent) FeMoco in NMF and in NMF-DMF mixtures is [Mo2Fe12S12]4-, but that other small organic anions such as NMF- may be present.

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 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.

Comparison of redox and EPR properties of the molybdenum iron proteins of Clostridium pasteurianum and Azotobacter vinelandii nitrogenases.

Both heterologous crosses of the Clostridium pasteurianum and Azotobacter vinelandii nitrogenase components are completely inactive, although the reasons for this incompatibility are not known. We have compared a number of properties of the MoFe proteins from these organisms (Cp1 and Av1, respectively) in an attempt to find differences that could explain this lack of functional activity. Optical and CD spectroscopic titrations are similar for both Av1 and Cp1, but EPR titrations are significantly different, suggesting different chemical reactivity patterns and/or magnetic interaction behavior. Similarly, reduction measurements on the six-electron-oxidized state of Cp1 and Av1 at controlled potentials indicate a difference in both the relative reduction sequence of the redox centers and the numerical values for their measured midpoint potentials. EPR measurements as a function of temperature also demonstrate that the relaxation behavior of the S = 3/2 MoFe centers associated with the proteins differ markedly. The Cp1 EPR signal only begins to undergo broadening above 65 K, whereas the Av1 signal is severely broadened above 25 K. These variations in the EPR properties for the two proteins are not likely to be due to differences in the stoichiometry and/or geometry of the MoFe cluster units themselves since similar EPR studies of the isolated cofactors showed them to be essentially identical. Thus, the different EPR behavior of the two proteins seems to arise either from protein constraints imposed on identical cofactors, and/or from magnetic interactions due to neighboring metal clusters.

Further characterization of the redox and spectroscopic properties of Azotobacter vinelandii ferritin.

Bacterial ferritin from Azotobacter vinelandii (AVBF) has many properties in common with and a number of properties distinct from the more thoroughly studied animal ferritins. The most notable differences are the high phosphate content of the mineral core and the presence of heme (12 per AVBF) in AVBF. In both ferritin types, redox reactions are essential to the iron release and deposition function of the ferritins. The heme reduction potential in apo AVBF is pH independent as are both the heme and core reduction potential in holo AVBF. pH measurements confirm the pH independence for heme reduction in apo AVBF; however, they establish the conflicting result that 1.7 +/- 0.2 protons per iron atom are taken up during core reduction. These results are interpreted as a two-step reduction process consisting of a pH independent reduction of heme in holo AVBF followed by a pH dependent reduction of the mineral core. Detailed spectroscopic studies have been undertaken to determine if heme-core interactions are detectable during the redox reactions of AVBF. Optical spectroscopy of the heme groups in apo AVBF demonstrate that all twelve are identical and undergo uniform and rapid reduction. EPR spectroscopy establishes the presence of both low-symmetry, g = 4.3, Fe3+ from the mineral core and low-spin heme with g values of 2.87, 2.32, and 1.46 in holo and identical g values for the low-spin heme in apo AVBF. EPR integration of the heme groups in both apo and holo gave values of 13.2 +/- 1.3 heme spins per AVBF at 4.2, 10, 25, 35, and 45 K. No heme perturbations were detected in holo or apo AVBF by Resonance Raman and circular dichroism spectroscopy. Both reduced and oxidized apo AVBF gave normal fluorescence emission at 330-340 nm when excited at 279 nm. These spectroscopic, redox, and reactivity results provide more detailed properties of AVBF for comparison with other bacterial and animal ferritins.

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.

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.

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.

A microcalorimetric procedure for evaluating the kinetic parameters of enzyme-catalyzed reactions: kinetic measurements of the nitrogenase system.

The mechanism of nitrogenase catalysis, as evaluated from steady-state kinetic measurements, is presently unresolved primarily due to conflicting results regarding the reaction order of the nitrogenase reductant, S2O2-4, at high concentrations. A microcalorimetric method was developed and is described which measures the rate of heat production (and hence the rate of reactant disappearance or product formation) as a function of time. Because each substrate reaction order has a unique profile for the rate of heat production with time, the described procedure provides a means for establishing the substrate reaction order for the enzyme-catalyzed reaction under consideration by visual inspection of the resulting thermogram. The rate constant and other kinetic parameters are obtained from analysis of the shape of the thermogram and thermodynamic parameters are evaluated from either the shape of or the area bound by the thermogram. Application of this procedure to the nitrogenase system has confirmed one-half- and first-order reaction orders under limiting conditions for the S2O2-4 and MgATP substrates during the enzyme-catalyzed reaction for this important biological process. From a single thermogram, the enthalpy of reaction and the kinetic rate law are readily evaluated. The procedure is completely general in nature and is applicable to any chemical or biochemical system that evolves heat.

Kinetics of dithionite ion utilization and ATP hydrolysis for reactions catalyzed by the nitrogenase complex from Azotobacter vinelandii.

The kinetics of S2O42-utilization and ATP hydrolysis during the nitrogenase-catalyzed H2 evolution and acetylene and nitrogen-reducing reactions were studied using a polarographic technique to monitor-S2O42-concentration. Rate constants for both S2O42-utilization and ATP hydrolysis were determined as a function of temperature and corresponding activation energies determined. The activation energy for ATP hydrolysis differs from that for product formation or S2O42-utilization by 5 kcal/mol above 20 degrees C and by 25 kcal/mol below 20 degrees C. The rate law for S2O42-utilization was determined and describes the enzyme catalyzed rate over a 1000-fold variation in S2O42-concentration and at least a 100-fold change in ATP concentration. The rate law for S2O42-utilization under N2-reducing conditions at 25 degrees C is given by -d([S2O42-]/dt = (2.3 x 10(-3) ET[S2O42-]1/2-[ATP]2)/([ATP]2 + K1[ATP] + K2), where ET is total enzyme concentration in mg/ml and K1 and K2 are equilibrium constants for ATP binding to nitrogenase. The half-order dependence of the rate on S2O42-concentration in interpreted in terms of the equilibrium S2O42- = 2SO2-, in which SO2- is the actual electron donor to nitrogenase. A partial mechanism incorporating these results is presented.

A chemical preparation of pure reduced viologens for use as biomolecular reducing reagents.

A chemical method is reported for conveniently preparing a variety of pure, reduced low-potential viologens for use as biomolecular reductants. The free radical, semiquinone viologen form is prepared anaerobically in aqueous solution by coproportionation of the dihydroviologen with the fully oxidized viologen according to the following reaction, using methyl viologen (MV) as an example: MV + MVH2 = 2 MV.. By varying the substituents on the viologen nitrogen atoms, a series of viologens of varying charge and reduction potential is easily obtained. Applications involving the reduction of various metalloproteins are presented.

H2-uptake activity of the MoFe protein component of Azotobacter vinelandii nitrogenase.

The MoFe protein from Azotobacter vinelandii catalyzes the reduction of methylene blue and other oxidants by H2 under anaerobic conditions. H2 uptake followed manometrically or by 3H2 transfer from the gas to aqueous phase occurs concomitantly with methylene blue disappearance monitored optically or coulometrically. The stoichiometry was found to be 1:1 methylene blue/H2. MoFe protein oxidized by transfer of approximately 4 e- seems to be the redox state of the protein most active in the catalytic step, although both the S2O4(2-)-reduced and 6-e- oxidized state have been shown to react, but at a much lower rate. The presence of H2 in the atmosphere above the MoFe protein offers increased protection against O2 inactivation.

Stoichiometry and spectral properties of the MoFe cofactor and noncofactor redox centers in the MoFe protein of nitrogenase from Azotobacter vinelandii.

Reductive EPR and optical titrations of oxidized MoFe protein using reduced methyl viologen as reductant were used to quantitate the stoichiometry of the various spectroscopically and electrochemically distinct redox centers in the oxidized MoFe protein. Three centers were found to correlate with the EPR signal development (MoFe cofactor centers), and three centers were found to be independent of the EPR signal (P clusters) but to demonstrate distinct optical and kinetic properties. Oxidative EPR and optical titrations of reduced MoFe protein are reported which support the presence of three P-cluster centers. The optical titrations show a distinct change in kinetic behavior between the MoFe cofactor and P-cluster centers. Controlled potential coulometry demonstrates that incremental oxidation of reduced protein by methylene blue, thionine, or indigodisulfonate occurs specifically at three P-cluster sites. Subsequent oxidation by methylene blue and thionine (but not indigodisulfonate) causes the EPR signal to disappear. Three P-cluster sites, two EPR sites, and one presently uncharacterized site are suggested by the results of this study.