Respiratory nitrate reductase
from
the denitrifying bacterium Pseudomonas stutzeri is an
iron-sulfur
enzyme containing the molybdenum cofactor. Hydrolysis of native nitrate
reductase with aqueous sulfuric acid revealed 0.92 mol of 5'-GMP per
mol
of enzyme. The pterin present in the molybdenum cofactor was liberated
from the protein and reacted with iodoacetamide. The resulting
di(carboxamidomethyl)
(cam) derivative was purified on a C18-cartridge
and
analyzed for its structural elements. Treatment of the cam derivative
with
nucleotide pyrophosphatase and subsequent HPLC analysis revealed the
formation
of di(cam)molybdopterin and 5'-GMP at a 1:1 molar ratio and with a
yield
of 79% with respect to the molybdenum content of the enzyme. Treatment
of the cam derivative with nucleotide pyrophosphatase and alkaline
phosphatase
led to the liberation of 0.51 mol dephosphodi(cam)molybdopterin and of
0.59 mol guanosine per mol of enzyme, which is equal to a molar ratio
of
1:1.2. The results indicate, that the organic moiety of the molybdenum
cofactor of nitrate reductase from P. stutzeri is molybdopterin
guanine dinucleotide of which one mol is contained per mol of nitrate
reductase.