Nitrogenase reactivity: effects of pH on substrate reduction and CO inhibition.
Molybdenum nitrogenase is composed of two separately purified proteins designated the iron protein (Fe protein) and the molybdenum-iron protein (MoFe protein), with the latter containing the substrate reduction site which is a metal cluster designated the iron-molybdenum cofactor (FeMo cofactor). In addition to its physiological substrates H+ and N2, nitrogenase reduces a number of nonphysiological substrates (e.g. C2H2 and N3-) and interacts with a number of similar molecules (e.g. CH3NC and CO) that serve as specific inhibitors. Despite their great diversity, all substrates are reduced by multiples of two electrons and require equivalent numbers of electrons and protons. Although the electron donor to a substrate is believed to be FeMo cofactor, the nature of the proton donor is unknown and might be different for different substrates. Here we report a three-component buffer assay system that eliminates variables of buffer type, ionic strength, and ATP and reductant availability and that is compatible with the nitrogenase system in the pH range 5.0-9.8. Preincubated studies and studies of the effects of pH on H2 evolution under Ar, H2 evolution under N2, H2 evolution under CO, and C2H2 reduction show that there is a group with a pK of ca. 6.3 that must be deprotonated for substrate reduction to occur and that there is a group with a pK of ca. 9.0 that must be protonated for substrate reduction to occur.(ABSTRACT TRUNCATED AT 250 WORDS)
Pham
D N
DN
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
Burgess
B K
BK
D000114:Acetylene / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D016948:Azotobacter vinelandii / Q000201:enzymology*; D002021:Buffers; D002248:Carbon Monoxide / Q000494:pharmacology*; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007501:Iron; D008982:Molybdenum; D009584:Nitrogen / Q000378:metabolism / Q000494:pharmacology; D009591:Nitrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009994:Osmolar Concentration; D010084:Oxidation-Reduction
D002021:Buffers; D002248:Carbon Monoxide; D006859:Hydrogen; D008982:Molybdenum; D000255:Adenosine Triphosphate; D007501:Iron; D009591:Nitrogenase; D009584:Nitrogen; D000114:Acetylene