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.

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.

A novel buffer system for separation of metal cations by capillary electrophoresis with indirect UV detection.

Generally, the buffers used for metal ion separations in capillary electrophoresis (CE) consist of a UV-active substance, pH-adjuster, and weak complexing reagent. This paper describes the successful separation of metal ions with a new buffer that contains no complexing reagent. Of several weakly basic compounds tested, 2-aminopyridine was selected as the most useful UV-active substance. It was used at a concentration of 15 mM with pH adjusted to 5.0 +/- 0.1 by acetic acid. The degree of protonation of the UV-active substance played an important role in detection. The stacking phenomenon was a significant contributor to efficiency in this buffer system, and water-diluted samples gave especially high efficiencies. When a 75-micron-i.d. fused-silica capillary was used, a separation efficiency of 1.8 x 10(5) was observed. Quantitative determinations of Ca2+, Mn2+, Zn2+, and Cd2+ were achieved with good linear calibration curves over the range of concentration from a few milligrams per liter to 100 mg/L. The detection limits were 0.2 mg/L for Ca2+, 0.4 mg/L for Mn2+ and Zn2+, and 0.6 mg/L for Cd2+, based on three times the baseline noise.

A microassay for serum dialyzable calcium and magnesium by ion chromatography.

We describe a method for determining dialyzable calcium and dialyzable magnesium in 50-microliter volumes of routinely handled aerobic serum. Calcium and magnesium concentrations were measured by ion chromatography after a 1-min equilibrium dialysis. Day-to-day precision (coefficient of variation, CV) was 3.8% for dialyzable calcium and 4.5% for dialyzable magnesium. Dilution of serum with water 1:1 as well as storage at 4 or -20 degrees C had no significant effects. Reference intervals for dialyzable calcium (58.1-68.1 mg/l) and magnesium (12.3-17.1 mg/l) were determined for 26 healthy adults. Lack of interference and the ability to determine simultaneously several analytes on a single specimen are advantages of the chromatographic method.

Comparison of serum total calcium, dialyzable calcium, and dialyzable magnesium in well and sick neonates.

We studied the relation of serum total calcium, dialyzable calcium, and dialyzable magnesium in 61 well and sick newborn infants aged 7-76 h. The infants' serum total calcium, dialyzable calcium and magnesium concentrations (ion chromatography method) were studied in comparison with the infant's history and sickness scale. We found that serum total calcium and dialyzable magnesium were lower in sick infants compared to well infants. Both serum total and dialyzable calcium concentrations initially decreased and then increased by about 30 h of age. Serum dialyzable magnesium concentrations increased with our infants' age. Serum total calcium values correlated significantly with the infant's birth weight, gestational age, 1-min Apgar score, respiratory distress, severity of sickness, serum bilirubin and sodium concentrations.