Redox and functional analysis of the Rieske ferredoxin component of the toluene 4-monooxygenase.
Toluene 4-monooxygenase catalyzes the NADH- and O2-dependent hydroxylation of toluene to form p-cresol. The four-protein complex consists of a diiron hydroxylase, an oxidoreductase, a catalytic effector protein, and a Rieske-type ferredoxin (T4moC). Phylogenetic analysis suggests that T4moC is part of a clade specialized for reaction with diiron hydroxylases, possibly reflected in the conservation of W69, whose indole side chain makes close contacts with a bridging sulfide. In order to further investigate the possible origins of this specialization, T4moC, mutated variants of T4moC, and three other purified ferredoxins (the Thermus Rieske protein, the Burkholderia cepacia Rieske-type biphenyl dioxygenase ferredoxin BphF, and the Ralstonia pickettii PK01 toluene monooxygenase TbuB, the Rieske-type ferredoxin from another diiron monooxygenase complex) were studied by redox potential measurements and their ability to complement the catalytic function of the reconstituted toluene 4-monooxygenase complex. A saturation mutagenesis of T4moC W69 indicates that an aromatic residue may modulate the redox potential and is also necessary for activity and/or stability. The redox potential of T4moC was determined to be -173 mV, W69F T4moC was -139 mV, and TbuB was -150 mV. For comparison, BphF had a redox potential of -157 mV [Couture et al. (2001) Biochemistry 40, 84-92]. Of these ferredoxins, all except BphF were able to provide catalytic activity. Given the range in redox potentials observed in the active ferredoxins, shape and electrostatics are strongly implicated in the catalytic specialization. Mutagenesis of other T4moC surface residues gave further insight into possible origins of catalytic specialization. Thus R65A T4moC gave an alteration in apparent KM only, while D82A/D83A T4moC gave alterations in both apparent kcat and KM. Since the different catalytic results were obtained by mutagenesis of residues lying on different sides of the protein adjacent to the [2Fe-2S] cluster, the results suggest that two different faces of T4moC may be involved in protein-protein interactions during catalysis.
Elsen
Nathaniel L
NL
Department of Biochemistry and Center for Eukaryotic Structural Genomics, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706-1544, USA.
Moe
Luke A
LA
McMartin
Lea A
LA
Fox
Brian G
BG
D000595:Amino Acid Sequence; D016956:Burkholderia cepacia / Q000201:enzymology / Q000235:genetics; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D006860:Hydrogen Bonding; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D049920:Ralstonia pickettii / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D055672:Static Electricity; D016963:Thermus thermophilus / Q000201:enzymology / Q000235:genetics
D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; C037989:Rieske iron-sulfur protein; D010105:Oxygenases; C068610:toluene-4-monooxygenase; D014450:Electron Transport Complex III