Plastocyanin-cytochrome f interactions: the influence of hydrophobic patch mutations studied by NMR spectroscopy.
Transient complex formation between plastocyanin from Prochlorothrix hollandica and cytochrome f from Phormidium laminosum was investigated using nuclear magnetic resonance (NMR) spectroscopy. Binding curves derived from NMR titrations at 10 mM ionic strength reveal a 1:1 stoichiometry and a binding constant of 6 (+/-2) x 10(3) M(-1) for complex formation, 1 order of magnitude larger than that for the physiological plastocyanin-cytochrome f complex from Ph. laminosum. Chemical-shift perturbation mapping indicates that the hydrophobic patch of plastocyanin is involved in the complex interface. When the unusual hydrophobic patch residues of P. hollandica plastocyanin were reverted to the conserved residues found in most other plastocyanins (Y12G/P14L), the binding constant for the interaction with cytochrome f was unaffected. However, the chemical shift perturbation map was considerably different, and the size of the average perturbation decreased by 40%. The complexes of both the wild-type and double mutant plastocyanin with cytochrome f were sensitive to ionic strength, contrary to the physiological complex. The possible implications of these findings for the mechanism of transient complex formation are discussed.
Crowley
Peter B
PB
Gorlaeus Laboratories, Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
Vintonenko
Nadejda
N
Bullerjahn
George S
GS
Ubbink
Marcellus
M
D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D000458:Cyanobacteria / Q000201:enzymology / Q000235:genetics; D003580:Cytochromes / Q000737:chemistry* / Q000235:genetics; D045348:Cytochromes f; D005998:Glycine / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D007930:Leucine / Q000235:genetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010970:Plastocyanin / Q000737:chemistry* / Q000235:genetics; D019692:Prochlorothrix / Q000737:chemistry / Q000235:genetics; D011392:Proline / Q000235:genetics; D011522:Protons; D014443:Tyrosine / Q000235:genetics
D003580:Cytochromes; D046911:Macromolecular Substances; D011522:Protons; D014443:Tyrosine; D006639:Histidine; D010970:Plastocyanin; D045348:Cytochromes f; D011392:Proline; D007930:Leucine; D005998:Glycine