Steady-state and time-resolved fluorescence studies on wild type and mutant Chromatium vinosum high potential iron proteins: Holo- and apo-forms

Detailed circular dichroism (CID), steady-state and time-resolved tryptopha n fluorescence studies on the holo- and apo- forms of high potential iron p rotein (HiPIP) from Chromatium vinosum and its mutant protein have been car ried out to investigate conformational properties of the protein. CD studie s showed that the protein does not have any significant secondary structure elements in the holo- or apo- HiPIP, indicating that the metal cluster doe s not have any effect on formation of secondary structure in the protein. S teady-state fluorescence quenching studies however, suggested that removal of the iron-sulfur ([Fe4S4](3+)) cluster from the protein leads to an incre ase in the solvent accessibility of tryptophans, indicating change in the t ertiary structure of the protein. CD studies on the holo- and apo- HiPIP al so showed that removal of the metal prosthetic group drastically affects th e tertiary structure of the protein. Time-resolved fluorescence decay of th e wild type protein was fitted to a four-exponentials model and that of the W80N mutant was fitted to a three-exponentials model. The time-resolved fl uorescence decay was also analyzed by maximum entropy method (MEM). The res ults of the MEM analysis agreed with those obtained from discrete exponenti als model analysis. Studies on the wild type and mutants helped to assign t he fast picosecond lifetime component to the W80 residue, which exhibits fa st fluorescence energy transfer to the [Fe4S4](3+) cluster of the protein. Decay-associated fluorescence spectra of each tryptophan residues were calc ulated from the time-resolved fluorescence results at different emission wa velengths. The results suggested that W80 is in the hydrophobic core of the protein, but W60 and W76 are partially or completely exposed to the solven t.