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.
Wilson
P E
PE
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84604, USA.
Bunker
J
J
Lowery
T J
TJ
Watt
G D
GD
D000227:Adenine Nucleotides / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000465:Algorithms; D016948:Azotobacter vinelandii / Q000201:enzymology*; D002021:Buffers; D004227:Dithionite / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D013696:Temperature; D013816:Thermodynamics
D000227:Adenine Nucleotides; D002021:Buffers; D004227:Dithionite; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; C029130:nitrogenase reductase