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Journal of Bacteriology, October 2008, p. 6493-6500, Vol. 190, No. 19
0021-9193/08/$08.00+0     doi:10.1128/JB.00790-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Jörg Johannes,1,{dagger} Alexander Bluschke,1 Nico Jehmlich,2 Martin von Bergen,2 and Matthias Boll1*

Institute of Biochemistry, University of Leipzig, Brüderstraβe 34, 04103 Leipzig, Germany,1 Department for Proteomics, Helmholtz Center for Environmental Research, Permoserstraβe 15, 04318 Leipzig, Germany2

Received 5 June 2008/ Accepted 21 July 2008

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 {alpha}{alpha}'β2 subunit composition, with {alpha} and {alpha}' representing isoforms of the flavin adenine dinucleotide (FAD)-containing subunit and β representing a c-type cytochrome. Fluorescence and mass spectrometric analysis suggested that one FAD was covalently linked to Tyr394 of the {alpha} subunit. In contrast, the {alpha}' 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 Km values (1.7 and 2.7 µM, 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 Kic (competitive inhibition) value of 18 ± 9 µM and a Kiu (uncompetitive inhibition) value of 235 ± 20 µM. A putative functional model for an unusual PCMH enzyme is presented.

* Corresponding author. Mailing address: Institute of Biochemistry, University of Leipzig, Brüderstraβe 34, 04103 Leipzig, Germany. Phone: 49-341-9736996. Fax: 49-341-9736919. E-mail: boll{at}uni-leipzig.de

{triangledown} Published ahead of print on 25 July 2008.

{dagger} Present address: Helmholtz Center Munich, Institute of Groundwater Ecology, Lugolstädter Landstr. 1, 85764 Neuherberg, Germany.

Journal of Bacteriology, October 2008, p. 6493-6500, Vol. 190, No. 19
0021-9193/08/$08.00+0     doi:10.1128/JB.00790-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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