Factors which stabilize the methylamine dehydrogenase-amicyanin electron transfer protein complex revealed by site-directed mutagenesis.
Methylamine dehydrogenase (MADH) and amicyanin form a physiologic complex within which electrons are transferred from the tryptophan tryptophylquinone (TTQ) cofactor of MADH to the type 1 copper of amicyanin. Interactions responsible for complex formation may be inferred from the crystal structures of complexes of these proteins. Site-directed mutagenesis has been performed to probe the roles of specific amino acid residues of amicyanin in stabilizing the MADH-amicyanin complex and determining the observed ionic strength dependence of complex formation. Conversion of Phe97 to Glu severely disrupted binding, establishing the importance of hydrophobic interactions involving this residue. Conversion of Arg99 to either Asp or to Leu increased the Kd for complex formation by 2 orders of magnitude at low ionic strength, establishing the importance of ionic interactions which were inferred from the crystal structure involving Arg99. Conversion of Lys68 to Ala did not disrupt binding at low ionic strength, but it did greatly diminish the observed ionic strength dependence of complex formation that is seen with wild-type amicyanin. These results demonstrate that the physiologic interaction between MADH and amicyanin is stabilized by a combination of ionic and van der Waals interactions and that individual amino acid residues on the protein surface are able to dictate specific interactions between these soluble redox proteins. These results also indicate that the orientation of MADH and amicyanin when they react with each other in solution is the same as the orientation of the proteins which is seen in the structure of the crystallized protein complex.
Davidson
V L
VL
Department of Biochemistry, The University of Mississippi Medical Center, Jackson, Mississippi 39216-4505, USA. davidson@fiona.umsmed.edu
Jones
L H
LH
Graichen
M E
ME
Mathews
F S
FS
Hosler
J P
JP
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism; D004926:Escherichia coli; D018698:Glutamic Acid; D045563:Indolequinones; D008239:Lysine; D008667:Metalloproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010231:Paracoccus denitrificans / Q000201:enzymology; D010649:Phenylalanine; D011487:Protein Conformation; D011809:Quinones / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000031:analogs & derivatives / Q000378:metabolism
D001426:Bacterial Proteins; D045563:Indolequinones; D008667:Metalloproteins; D011809:Quinones; D011994:Recombinant Proteins; C017079:mauC protein, Methylobacterium extorquens; C070569:tryptophan tryptophylquinone; D018698:Glutamic Acid; D010649:Phenylalanine; D003300:Copper; D014364:Tryptophan; D001120:Arginine; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors; D008239:Lysine