Redox potential of the cytochrome <i>c</i> in the flavocytochrome <i>p</i>‐cresol methylhydroxylase

Hopper, David J.

doi:10.1016/0014-5793(83)80738-8

Cited by 24 publications

(16 citation statements)

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“…In this respect, the enzyme is similar to other PCMHs, although slightly larger than most (9). The midpoint redox potential of +232 mV also lies within the range of those reported for the PCMHs from pseudomonads, which range from +226 mV to +250 mV (8). The pattern of the steady-state kinetics and inhibition by a high concentration of phenazine methosulfate, the electron acceptor in the assay, were also similar to those reported for PCMH A from P. putida NCIB 9869.…”

Section: Discussionsupporting

confidence: 81%

“…One answer to this is the p-cresol methylhydroxylase (PCMH) that has been isolated from aerobically grown Pseudomonas putida, which is thought to act by dehydrogenation of the substrate to give a quinone methide that is then hydrated (6,7). The enzyme is a flavocytochrome c consisting of two flavoprotein subunits, each of Mr 49,000, and two cytochrome c subunits, each of Mr 8,800. The flavin is covalently bound to the protein through a tyrosine residue (13).…”

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confidence: 99%

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p-cresol methylhydroxylase from a denitrifying bacterium involved in anaerobic degradation of p-cresol

1991

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A bacterium, strain PC-07, previously isolated as part of a coculture capable of growing on p-cresol under anaerobic conditions with nitrate as the acceptor was identified as an Achromobacter sp. The first enzyme of the pathway, p-cresol methylhydroxylase, which converts its substrate into p-hydroxybenzyl alcohol, was purified.The enzyme had an Mr of 130,000 and the spectrum of a flavocytochrome. It was composed of flavoprotein subunits of Mr 54,000 and cytochrome subunits of Mr 12,500. The midpoint redox potential of the cytochrome was 232 mV. The-Km and kcat for p-cresol were 21 ,uM and 112 s-1 respectively, and the Km for phenazine methosulfate, the artificial acceptor used ig the assays, was determined to be 1.7 mM. These properties place the enzyme in the same class as the p-cresol methylhydroxylases from aerobically isolated Pseudomonas spp.

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Section: Discussionsupporting

confidence: 81%

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confidence: 99%

p-cresol methylhydroxylase from a denitrifying bacterium involved in anaerobic degradation of p-cresol

1991

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“…The genes from P. putida NCIMB 9869 were for the plasmid-encoded A form of PCMH, and the genes from P. putida NCIMB 9866 were also plasmid encoded. The PCMH has been isolated from several Pseudomonas species and other microorganisms (24,25,28,33,41,51,57). A similar enzyme, 4-ethylphenol methylenehydroxylase from Pseudomonas putida JD1, has also been purified and studied (55).…”

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Cloning, sequencing, and expression of the structural genes for the cytochrome and flavoprotein subunits of p-cresol methylhydroxylase from two strains of Pseudomonas putida

et al. 1994

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The structural genes for the flavoprotein subunit and cytochrome c subunit of p-cresol (4-methylphenol) methyihydroxylase (PCMH) from Pseudomonas putida NCIMB 9869 (National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland) and P. putida NCIMB 9866 were cloned and sequenced. The genes from P. putida NCIMB 9869 were for the plasmid-encoded A form of PCMH, and the genes from P. putida NCIMB 9866 were also plasmid encoded. The PCMH has been isolated from several Pseudomonas species and other microorganisms (24,25,28,33,41,51,57). A similar enzyme, 4-ethylphenol methylenehydroxylase from Pseudomonas putida JD1, has also been purified and studied (55).The best-characterized enzyme from this group is PCMH69A, the plasmid-encoded A form of PCMH from P. putida NCIMB 9869 (National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, Scotland). Its 3-A (1 A = 0.1 nm) three-dimensional structure is known (42) stereochemistry of its oxidation ofp-cresol, 4-ethylphenol, and 1-(4'-hydroxyphenyl)ethanol (45,46). The flavoprotein and cytochrome subunits have been separated, characterized, and reconstituted into native enzyme (35,36). Further, the amino acid sequences of the entire cytochrome subunit and the N terminus and flavin attachment site of the flavoprotein of PCMH69A have been determined (44,48).In the absence of the complete amino acid sequence, several important features have been gleaned from the three-dimensional structure of PCMH69A. For example, the cytochrome subunit structure is similar to those of other c-type cytochromes, but the structure of the flavoprotein subunit is unlike that of any other known protein structure. It was found that the planes of the isoalloxazine and heme rings form a 650 angle, and a heme vinyl methylene carbon is -9 A from the 8ot-methyl group of the flavin (42). To obtain further detailed structural information from the crystallographic data, knowledge of the complete amino acid sequence of the flavoprotein is essential. These data are also important in our quest to better understand the mechanism of action of PCMH; in this regard, investigation of site-specifically mutated forms of PCMH will be extremely useful.P. putida NCIMB 9866 (hereafter referred to as P. putida 9866) produces a single form of PCMH (PCMH66), while P. putida NCIMB 9869 (hereafter referred to as P. putida 9869) manufactures an A and a B form of the enzyme. The genes for 6349 on May 9, 2018 by guest

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“…The latter is further converted to 4-hydroxybenzaldehyde by the same enzyme, p-cresol-methylhydroxylase (13,22). The standard redox potential (E 0 Ј) of the couple 4-hydroxybenzylalcohol/p-cresol is in the range of ϩ80 mV (calculated as described before [42]), and the methylhydroxylase reaction is coupled with the reduction of a c-type cytochrome with a midpoint potential of around ϩ230 mV (21,22).It has been proposed that sulfate-reducing bacteria degrade p-cresol via methyl group oxidation, too (19,28,41). This would be surprising, since release of electrons at the redox potentials mentioned above would be difficult for these bacteria: transfer of electrons from the reduced cytochrome of pcresol-methylhydroxylase to either adenosine 5Ј-phosphosulfate (E 0 Ј ϭ Ϫ60 mV [42]) or sulfite (E 0 Ј ϭ Ϫ116 mV [42]) as the electron acceptor would require a substantial energy input.…”

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confidence: 99%

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confidence: 99%

Initiation of Anaerobic Degradation of p -Cresol by Formation of 4-Hydroxybenzylsuccinate in Desulfobacterium cetonicum

et al. 2001

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The anaerobic bacterium Desulfobacterium cetonicum oxidized p-cresol completely to CO 2 with sulfate as the electron acceptor. During growth, 4-hydroxybenzylsuccinate accumulated in the medium. This finding indicated that the methyl group of p-cresol is activated by addition to fumarate, analogous to anaerobic toluene, m-xylene, and m-cresol degradation. In cell extracts, the formation of 4-hydroxybenzylsuccinate from p-cresol and fumarate was detected at an initial rate of 0.57 nmol min ؊1 (mg of protein) ؊1. This activity was specific for extracts of p-cresol-grown cells. 4-Hydroxybenzylsuccinate was degraded further to 4-hydroxybenzoylcoenzyme A (CoA), most likely via ␤-oxidation. 4-Hydroxybenzoyl-CoA was reductively dehydroxylated to benzoyl-CoA. There was no evidence of degradation of p-cresol via methyl group oxidation by p-cresol-methylhydroxylase in this bacterium.The toxic aromatic compound p-cresol (4-methylphenol) is a constituent of disinfectants and preservatives and is used largely in the formulation of antioxidants and in the fragrance and dye industries (1). It originates mainly from coal gasification plants, fractionation of coal tar, and a variety of synthetic processes. p-Cresol is also formed from tyrosine by several anaerobic bacteria (16,41,44). Anaerobic degradation of pcresol could be demonstrated with pure cultures of nitratereducing, iron-reducing, and sulfate-reducing bacteria and under methanogenic conditions (2,7,19,25,29,37,39,43). It has been well established that denitrifying bacteria metabolize pcresol, cognate to its degradation by aerobic bacteria (13 and references therein), through a sequence of oxidation reactions leading to 4-hydroxybenzoate, with water as the oxygen source (7,14,34). In the initial step, the methyl group of p-cresol is enzymatically oxidized, probably via formation of a quinone methide intermediate (13,20), to form 4-hydroxybenzylalcohol. The latter is further converted to 4-hydroxybenzaldehyde by the same enzyme, p-cresol-methylhydroxylase (13,22). The standard redox potential (E 0 Ј) of the couple 4-hydroxybenzylalcohol/p-cresol is in the range of ϩ80 mV (calculated as described before [42]), and the methylhydroxylase reaction is coupled with the reduction of a c-type cytochrome with a midpoint potential of around ϩ230 mV (21,22).It has been proposed that sulfate-reducing bacteria degrade p-cresol via methyl group oxidation, too (19,28,41). This would be surprising, since release of electrons at the redox potentials mentioned above would be difficult for these bacteria: transfer of electrons from the reduced cytochrome of pcresol-methylhydroxylase to either adenosine 5Ј-phosphosulfate (E 0 Ј ϭ Ϫ60 mV [42]) or sulfite (E 0 Ј ϭ Ϫ116 mV [42]) as the electron acceptor would require a substantial energy input.In the present study, we employed in vitro assays and physiological studies to examine sulfidogenic p-cresol degradation by Desulfobacterium cetonicum. With this bacterium, it was shown recently that anaerobic degradation of m-cresol proceeded via additi...

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Redox potential of the cytochrome c in the flavocytochrome p‐cresol methylhydroxylase

Cited by 24 publications

References 9 publications

p-cresol methylhydroxylase from a denitrifying bacterium involved in anaerobic degradation of p-cresol

p-cresol methylhydroxylase from a denitrifying bacterium involved in anaerobic degradation of p-cresol

Cloning, sequencing, and expression of the structural genes for the cytochrome and flavoprotein subunits of p-cresol methylhydroxylase from two strains of Pseudomonas putida

Initiation of Anaerobic Degradation of p -Cresol by Formation of 4-Hydroxybenzylsuccinate in Desulfobacterium cetonicum

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