Unfolding the unique c-type heme protein, Chlamydomonas reinhardtii cytochrome f.
We have studied the unfolding reaction of cytochrome f from the green alga Chlamydomonas reinhardtii. Cytochrome f is different from all other c-type heme proteins in that it is a large, two-domain protein with predominantly beta-sheet structure. Moreover, the sixth axial ligand to the heme-iron is unique in cytochrome f: it is provided by the N-terminal alpha-amino group. Unfolding of oxidized and reduced cytochrome f by guanidine hydrochloride (GuHCl) was monitored by far-UV circular dichroism (CD), Soret absorption, and tyrosine emission: the same unfolding curves were obtained regardless of method. Neither oxidized nor reduced unfolded cytochrome f can be refolded at neutral pH. At pH 3.5 refolding takes place (upon dilution to lower denaturant concentrations or by electron injection to the unfolded, oxidized form), although the reaction is extremely slow. Reduced cytochrome f appears much more resistant towards denaturant perturbation than the oxidized form (in pH range 7-3.5). The heme in unfolded cytochrome f remains low-spin to pH 4 but turns high-spin at pH 3.5 (presumably due to protonation of the N-terminal amino group). Our results suggest that the unfolding process for cytochrome f is complex, involving kinetically trapped intermediates not resolvable by spectroscopy.
Sabahi
Ali
A
Chemistry Department, Tulane University, 6832 St. Charles Avenue, New Orleans, LA 70118-5698, USA.
Wittung-Stafshede
Pernilla
P
D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D002942:Circular Dichroism; D003580:Cytochromes / Q000737:chemistry*; D045348:Cytochromes f; D004795:Enzyme Stability; D019791:Guanidine; D006420:Hemeproteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D014443:Tyrosine / Q000737:chemistry
D003580:Cytochromes; D006420:Hemeproteins; D014443:Tyrosine; D045348:Cytochromes f; D019791:Guanidine