One Molecule of Molybdopterin Guanine Dinucleotide is Associated with Each Subunit of the Heterodimeric Mo‐Fe‐S Protein Transhydroxylase of Pelobacter acidigallici as Determined by SDS/PAGE and Mass Spectrometry

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Abstract
The molybdenum‐containing iron‐sulfur protein 1,2,3,5‐tetrahydroxybenzene:1,2,3‐trihydroxyben‐zene hydroxyltransferase (transhydroxylase) of Pelobacter acidigallici was investigated by various techniques including mass spectrometry and electron paramagnetic resonance. Mass spectrometry confirmed that the 133‐kDa protein is a heterodimer consisting of an α subunit (100.4 kDa) and a β subunit (31.3 kDa). The presence of a molybdenum cofactor was documented by fluorimetric analysis of the oxidized form A of molybdopterin. The enzyme contained 1.55 ± 0.14 mol pterin and 0.92 ± 0.25 mol molybdenum/mol enzyme (133 kDa). Alkylation of the molybdenum cofactor with iodoacetamide formed di(carboxamidomethyl)‐molybdopterin. Upon acid hydrolysis, 1.4 mol 5′GMP/mol enzyme (133 kDa) was released indicating that molybdenum is bound by a molybdopterin guanine dinucleotide. The α and β subunits were separated by preparative gel electrophoresis. Both subunit fractions were free of molybdenum but contained equal amounts of a fluorescent form of the molybdenum cofactor. Mass spectrometry at various pH values revealed that an acid‐labile cofactor was released from the large subunit and also from the small subunit. At X‐band, 5–25 K, transhydroxylase (as isolated) showed minor EPR resonances with apparent g values around 4.3, 2.03 and, depending on the preparation, a further signal at g of approximately 1.98. This signal was still detectable above 70 K and was attributed to a Mo(V) center. Upon addition of dithionite, a complex set of intense resonances appeared in the region g 2.08–1.88. From their temperature dependence, three distinct sites could be identified: the Fe‐S center I with gx,y,z at approximately 1.875, 1.942 and 2.087 (gav 1.968, detectable 20K); and the Mo(V) center consisting of a multiple signal around g 1.98 (detectable >70 K).

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