Insight into covalent flavinylation and catalysis from redox, spectral, and kinetic analyses of the R474K mutant of the flavoprotein subunit of p-cresol methylhydroxylase.
Each flavoprotein subunit (PchF) of p-cresol methylhydroxylase (PCMH) has flavin adenine dinucleotide (FAD) covalently tethered to Tyr384. The PCMH structure suggests that Arg474 in PchF is required for self-catalytic covalent flavinylation and for substrate oxidation. The replacement of Arg474 with Lys was carried out to probe the subtleties of the role of Arg474 in these processes. In nearly all of the aspects examined, the mutant protein showed compromised properties relative to the wild-type protein, including the tenacity of noncovalent FAD binding to the apo-protein, the rate of covalent flavinylation, the affinity of the covalent flavoprotein for PchC (the cytochrome subunit), the k(cat) for substrate oxidation, and the affinity for substrate analogues in the formation of FAD-charge-transfer complexes (CT complexes). Nevertheless, because the mutant retains these attributes, the comparison allows for an examination of the role of this residue in the various properties of the enzyme. A correlation is proposed to exist between nu(m), the frequency for the absorbance maximum of the CT complex with a substrate analogue, and k(cat), the steady-state rate constant for oxidation of p-cresol by various forms of PCMH and PchF; both nu(m) and k(cat) can be expressed as functions of the ionization potential of the donor (I(D)) and the electron affinity of the acceptor (E(A)). This correlation is a better predictor of the rate constant for substrate oxidation than is the magnitude of the redox potential, E(m,7), of the bound FAD, which was determined for the various mutant enzyme species and compared with those of the wild type.
Efimov
Igor
I
Molecular Biology Division, Department of Veterans Affairs Medical Center, San Francisco, California 94121, USA.
Cronin
Ciarán N
CN
Bergmann
David J
DJ
Kuusk
Vladislav
V
McIntire
William S
WS
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017354:Point Mutation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016958:Pseudomonas putida / Q000201:enzymology
D001426:Bacterial Proteins; D005420:Flavoproteins; D021122:Protein Subunits; D005182:Flavin-Adenine Dinucleotide; D006899:Mixed Function Oxygenases; C049502:4-cresol dehydrogenase (hydroxylating)