Purification and Characterization of Active-Site Components of the Putative p -Cresol Methylhydroxylase Membrane Complex from Geobacter metallireducens

Abstract

p-Cresol methylhydroxylases (PCMH) from aerobic and facultatively anaerobic bacteria are soluble, periplasmic flavocytochromes that catalyze the first step in biological p-cresol degradation, the hydroxylation of the substrate with water. Recent results suggested that p-cresol degradation in the strictly anaerobic Geobacter metallireducens involves a tightly membrane-bound PCMH complex. In this work, the soluble components of this complex were purified and characterized. The data obtained suggest a molecular mass of 124 ؎ 15 kDa and a unique ␣␣␤ 2 subunit composition, with ␣ and ␣ representing isoforms of the flavin adenine dinucleotide (FAD)-containing subunit and ␤ representing a c-type cytochrome. Fluorescence and mass spectrometric analysis suggested that one FAD was covalently linked to Tyr 394 of the ␣ subunit. In contrast, the ␣ subunit did not contain any FAD cofactor and is therefore considered to be catalytically inactive. The UV/visible spectrum was typical for a flavocytochrome with two heme c cofactors and one FAD cofactor. p-Cresol reduced the FAD but only one of the two heme cofactors. PCMH catalyzed both the hydroxylation of p-cresol to p-hydroxybenzyl alcohol and the subsequent oxidation of the latter to p-hydroxybenzaldehyde in the presence of artificial electron acceptors. The very low K m values (1.7 and 2.7 M, respectively) suggest that the in vivo function of PCMH is to oxidize both p-cresol and p-hydroxybenzyl alcohol. The latter was a mixed inhibitor of p-cresol oxidation, with inhibition constants of a K ic (competitive inhibition) value of 18 ؎ 9 M and a K iu (uncompetitive inhibition) value of 235 ؎ 20 M. A putative functional model for an unusual PCMH enzyme is presented.

Anaerobic Activation of p -Cymene in Denitrifying Betaproteobacteria: Methyl Group Hydroxylation versus Addition to Fumarate

“…This is subsequently transformed to 4-hydroxybenzyl alcohol via a nucleophilic attack of a water molecule at the methide carbon atom, i.e., a formal 1,6-addition of water to the quinone-methide intermediate (25,68). Based on homology, a similar mechanism for p-cresol hydroxylation may be envisioned for Pch from G. metallireducens T (69). It may be assumed that the hydroxyl group plays an integral role in the reaction to allow formation of the quinone-methide intermediate based on electron donation either from one of the lone pairs of the hydroxyl group or from the corresponding oxyanion.…”

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

“…13). The purification and characterization of the soluble PcmIJ and PcmG components confirmed that p-cresol methylhydroxylase from G. metallireducens has a unique ␣␣Ј␤ 2 -subunit composition, with the ␣ subunit (PcmI) harboring the typical FAD cofactor, which is lacking in the catalytically inactive ␣Ј-subunit (PcmJ), and the ␤-subunit (PcmG) representing a c-type cytochrome (171). In contrast to other aerobic and facultatively anaerobic p-cresol-degrading organisms, G. metallireducens appears to use p-cresol methylhydroxylase for both p-cresol and p-hydroxybenzyl alcohol oxidation in vivo.…”

“…Although the oxidation of p-hydroxybenzyl alcohol to p-hydroxybenzaldehyde can also be carried out by p-cresol methylhydroxylase, an NAD ϩ -dependent p-hydroxybenzyl alcohol dehydrogenase was supposed to catalyze this reaction in vivo (75,171,183). This oxygen-independent pcresol hydroxylation has also been described for several denitrifying bacteria such as Thauera and Azoarcus strains (239,308) as well as for an Achromobacter strain (162).…”

“…Two vertical lines mean that the genes are not adjacent in the genome. (171,277). The existence of a p-cresol-induced membrane-bound cytochrome bc 1 -like complex, which usually mediates electron transfer between quinols and cytochrome c, could also account for the unusual asymmetric architecture of the p-cresol methylhydroxylase with two differing ␣-subunits.…”

“…The existence of a p-cresol-induced membrane-bound cytochrome bc 1 -like complex, which usually mediates electron transfer between quinols and cytochrome c, could also account for the unusual asymmetric architecture of the p-cresol methylhydroxylase with two differing ␣-subunits. Thus, it has been suggested that the noncatalytic ␣Ј-subunit may be involved in mediating both alternative electron transfer routes either to the periplasmic cytochrome c pool (in the case of p-cresol oxidation) or to the menaquinone pool (in the case of phydroxybenzyl alcohol oxidation), which would significantly increase the energy yield in an obligately anaerobic organism (171). The oxidation of the p-hydroxybenzaldehyde generated by the p-cresol methylhydroxylase to form 4-HBA is carried out by an NADP ϩ -dependent soluble dehydrogenase in G. metallireducens.…”