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

Abstract

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 a subunit (100.4 kDa) and a j? 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 1.55t0.14 mol pterin and 0.9220.25 mol molybdenundmol enzyme (133 kDa). Alkylation of the molybdenum cofactor with iodoacetamide formed di(carboxamidomethy1)-molybdopterin. Upon acid hydrolysis, 1.4 mol 5'GMP/mol enzyme (1 33 kDa) was released indicating that molybdenum is bound by a molybdopterin guanine dinucleotide. The a and j? 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 gn,y,z at approximately 1.875, 1.942 and 2.087 (gav 1.968, detectable <20 K); the Fe-S center I1 with g,,,, 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).

Towards the reaction mechanism of pyrogallol–phloroglucinol transhydroxylase of Pelobacter acidigallici

“…When enriched enzyme preparation was replaced by pure transhydroxylase in an assay with pyrogallol and tetramethylene sulfoxide, tetrahydroxybenzene was still formed, no matter whether 1% SDS was present or not. Since transhydroxylase is known to be rather insensitive towards SDS [8,9], this observation indicates that both reactions in Fig. 1 were carried out by the transhydroxylase enzyme.…”

“…Cyanide is not a potent inhibitor for our transhydroxylase [8], and it may not belong to this group, therefore. However, it does not group with other molybdenum enzymes either [9,12].…”

“…5). In these redox reactions, the iron sulfur clusters of the transhydroxylase enzyme [9] could be involved. The transhydroxylase reaction does not require external redox carriers in vitro.…”

“…Previous studies reported a formation of tetrahydroxybenzene from pyrogallol in the presence of high concentrations of dimethyl sulfoxide [1] indicating that tetrahydroxybenzene could be synthesized in vivo by a net hydroxylation of pyrogallol [6,7]. Similar to other hydroxylating enzymes, the pyrogallol^phloroglucinol transhydroxylase of P. acidigallici is a MoFeS protein, and contains iron sulfur centers and molybdopterin guanine dinucleotide as cofactors [8,9].…”

4-hydroxybenzoyl coenzyme A reductase (dehydroxylating) is required for anaerobic degradation of 4-hydroxybenzoate by Rhodopseudomonas palustris and shares features with molybdenum-containing hydroxylases

“…A molybdenum-containing transhydroxylase that converts pyrogallol to phloroglucinol, with the involvement of 1,2,3,5-tetrahydroxybenzene as a cosubstrate and coproduct, has been described. This enzyme, which is a heterodimer with two dissimilar subunits, is not sensitive to oxygen and is likely related to molybdenum-containing hydroxylases (40,41).…”