Recombinant Toluene-4-monooxygenase: Catalytic and Mössbauer Studies of the Purified Diiron and Rieske Components of a Four-Protein Complex
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- 作者：Jeremie D. Pikus ; Joey M. Studts ; Catalina Achim ; Karl E. Kauffmann ; Eckard Mü ; nck ; Robert J. Steffan ; Kevin McClay ; and Brian G. Fox
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：July 16, 1996
- 年：1996
- 卷：35
- 期：28
- 页码：9106 - 9119
- 全文大小：527K
- 年卷期：v.35,no.28(July 16, 1996)
- ISSN：1520-4995
文摘
Expression of the tmoA-F gene cluster fromPseudomonas mendocina KR1 inEscherichiacoli BL21(DE3) produces a catalytically active form of thetoluene-4-monooxygenase (T4MO) complex.Here we report the purification and characterization of foursoluble proteins required for the in vitroreconstitution of T4MO catalytic activity. These proteins are adiiron hydroxylase (T4MOH), a Rieske-type ferredoxin (T4MOC), an effector protein (T4MOD), and an NADHoxidoreductase (T4MOF). TheT4MOH component is composed of the tmoA, tmoB,and tmoE gene products [quaternary structure(
)2,Mr 
220 kDa]. The T4MOA polypeptidecontains two copies of the amino acid sequence motif (D/E)X(28-37)DEXRH; thesame motif provides all of the protein-derived ligands to the diironcenters ofribonucleotide reductase, the soluble methane monooxygenase, and thestearoyl-ACP 
9 desaturase.Mössbauer, optical, and EPR measurements show that the T4MOHcontains diiron centers and suggestthat the diiron center contains hydroxo bridge(s) in the diferricstate, as observed for methanemonooxygenase. Mössbauer and EPR measurements also show thatthe T4MOC contains a Rieske-typeiron-sulfur center. This assignment is in accord with thepresence of the amino acid sequence motifCPHX(15-17)CX2H,which has also been found in the bacterial, chloroplastic, andmitochondrial Rieskeproteins as well as the bacterial NADH-dependentcis-dihydrodiol-forming aromatic dioxygenases.Whilesingle-turnover catalytic studies confirm the function of the T4MOH asthe hydroxylase, the NADH-dependent multiple-turnover hydroxylation activity is increased by morethan 100-fold in the presence ofthe T4MOC, which mediates highly specific electron transfer between theT4MOF and the T4MOH.The T4MOD can be purified as an 11.6 kDa monomeric protein devoidof cofactors or redox-activemetal ions; this component is also detected as a substoichiometricconsitutent of the purified T4MOH.The rate of the hydroxylation reaction can be mildly stimulated bythe further addition of separatelypurified T4MOD to the T4MOH, implying the formation of a high affinity,catalytically competent complexbetween these two components. These characterizations define anovel, four-component oxygenasecombining elements from the soluble methane oxidation complex of themethanotrophic bacteria and thearomatic hydroxylation complexes of the soil pseudomonads.
)2,Mr 220 kDa]. The T4MOA polypeptidecontains two copies of the amino acid sequence motif (D/E)X(28-37)DEXRH; thesame motif provides all of the protein-derived ligands to the diironcenters ofribonucleotide reductase, the soluble methane monooxygenase, and thestearoyl-ACP 9 desaturase.Mössbauer, optical, and EPR measurements show that the T4MOHcontains diiron centers and suggestthat the diiron center contains hydroxo bridge(s) in the diferricstate, as observed for methanemonooxygenase. Mössbauer and EPR measurements also show thatthe T4MOC contains a Rieske-typeiron-sulfur center. This assignment is in accord with thepresence of the amino acid sequence motifCPHX(15-17)CX2H,which has also been found in the bacterial, chloroplastic, andmitochondrial Rieskeproteins as well as the bacterial NADH-dependentcis-dihydrodiol-forming aromatic dioxygenases.Whilesingle-turnover catalytic studies confirm the function of the T4MOH asthe hydroxylase, the NADH-dependent multiple-turnover hydroxylation activity is increased by morethan 100-fold in the presence ofthe T4MOC, which mediates highly specific electron transfer between theT4MOF and the T4MOH.The T4MOD can be purified as an 11.6 kDa monomeric protein devoidof cofactors or redox-activemetal ions; this component is also detected as a substoichiometricconsitutent of the purified T4MOH.The rate of the hydroxylation reaction can be mildly stimulated bythe further addition of separatelypurified T4MOD to the T4MOH, implying the formation of a high affinity,catalytically competent complexbetween these two components. These characterizations define anovel, four-component oxygenasecombining elements from the soluble methane oxidation complex of themethanotrophic bacteria and thearomatic hydroxylation complexes of the soil pseudomonads.