On the presence of a novel covalently bound oxidation-reduction cofactor, iron and labile sulfur in trimethylamine dehydrogenase

Abstract

Trimethylamine dehydrogenase from a facultative methylotroph contains 4 g atoms each of Fe and S and an unknown, covalently bound, yellow coenzyme. The absorbance of the enzyme in the visible range (λmax=445 nm) is extensively bleached by dithionite. Reduction by substrate causes less extensive bleaching and the appearance of a three banded spectrum which may be representative of a free radical form. Denaturation liberates the FeS center(s) but not the organic coenzyme. The latter is covalently linked to the protein via an amino acid residue and is solubilized on proteolytic digestion in the form of the peptide. The coenzyme-peptide has been purified to a constant ratio of amino acid to coenzyme. The oxidized and reduced forms show maximal absorbance at 437 nm and 380 nm respectively. Based on dithionite titrations its molar absorbance at 437 nm is 12,300 in the oxidized and 4000 in the dithionite reduced form. The cofactor is very labile to photolysis giving rise to several products the predominant one of which shows fluorescence excitation and emission maxima at 394 and 500 nm, respectively. After cleavage of the hydrolyzable amino acids in HCl, the compound consumed 3 moles of periodate. Digestion with aminopeptidase M yields a compound with a single amino acid and ∼1 mole of organic P present. Acid phosphatase, but not nucleotide pyrophosphatase affects its mobility. These findings suggest that the coenzyme-peptide is isolated in the form of a mononucleotide, containing a 5-carbon alcohol. The physical and chemical properties of the compound do not agree with those of known flavin or pyridoxine derivatives but are not incompatible with a covalently linked pteridine (lumazine) derivative, although no proof for such a structure is so far available.