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.
The molybdenum-containing iron-sulfur protein 1,2,3,5-tetrahydroxybenzene: 1,2,3-trihydroxybenzene 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 alpha subunit (100.4 kDa) and a beta 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 alpha and beta 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 cofactors. 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 < 20 K); the Fe-S center II with gx,y,z at approximately 1.872, 1.955 and 2.051 (gav 1.959, detectable > 20 K); and the Mo(V) center consisting of a multiple signal around g 1.98 (detectable > 70 K).
Reichenbecher
W
W
Fakultät für Biologie, Universität Konstanz, Germany.
Rüdiger
A
A
Kroneck
P M
PM
Schink
B
B
D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D003067:Coenzymes; D004578:Electron Spin Resonance Spectroscopy; D006150:Guanine Nucleotides / Q000737:chemistry*; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D013058:Mass Spectrometry; D008667:Metalloproteins / Q000737:chemistry; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D008982:Molybdenum / Q000378:metabolism*; D000090006:Molybdenum Cofactors; D011487:Protein Conformation; D011621:Pteridines / Q000737:chemistry; D011622:Pterins / Q000737:chemistry*
D003067:Coenzymes; D006150:Guanine Nucleotides; D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D006899:Mixed Function Oxygenases; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D011621:Pteridines; D011622:Pterins; C063761:molybdopterin guanine dinucleotide; C022261:molybdopterin