The goal of the project is to understand how iron sulfur proteins modulate reduction potential in order to perform specific functions.  The family of Rieske proteins is an ideal model to study, since it encompasses several proteins that have a wide range of reduction potentials (-150 mV to +475 mV) in a common protein fold.   The research plan has been to produce a series of mutants using the Rieske protein from Thermus thermophilus and characterizing each mutant using several spectroscopic and structural techniques. 

Three mutants of the Rieske protein from T. thermophilus have been produced: Y158F, L135A, and G156S.  Y158F is designed to remove a single hydrogen bond from the S_g of Cysteine to the cluster in order to evaluate the effect of the removal on reduction potential.  L135A is designed to test how the hydrophobic nature of the region encompassing the cluster affects potential.  G156S is a novel mutant not previously seen in the literature. It is designed to add a single hydrogen bond to the S* atom within the cluster.  All 3 mutants have been sequenced, and the mutation confirmed using mass spectrometry.  The mutants have been purified with yields of 15-30 mg mutant protein per 6L bacterial culture.  A new mutant has been designed, L135E, and will be made this semester, which will test the effect of adding a negative charge to the region of the cluster.

The wild type and the mutant proteins have also been subjected to crystallization.  A condition at low pH (~4.6) in PEG 400 was identified and the wild type, Y158F and L135A crystallize under the condition.  However, data taken on the crystals of the wild type resulted in structures in which the cluster was no longer intact, apparently degrading while subjected to the X-rays during the data acquisition.  Alternate crystallization conditions at a higher pH, yet still below the first pK_a of the protein, are being tested.  Several promising leads have produced low quality crystals, and refinement of the conditions is underway. 

G156S has been crystallized under a unique condition (sodium-potassium phosphate and HEPES pH 7.0) and produced high enough quality crystals to collect data locally at the UT Health Science Center in the laboratory of John Hart.  The resolution was 2.8Å and the space group P6₃22.  There are 4 proteins in the asymmetric unit.  Refinement of the structure is in progress.

The UV-Visible spectrum of each of the 3 produced mutants has been recorded and compared to the wild type.  In the wild type there are three bands, 328 nm, 458 nm, and a shoulder at 572 nm.  L135A is almost identical to the wild type, with just a small change in ε at these wavelengths.  Y158F has two red shifted peaks, 340 nm and 466 nm, but maintains the shoulder at 572 nm.  The spectrum of G156S is considerably different than the wild type.  It is interpreted as large blue shifts of each of the peaks, to 309 nm, 380 nm and 438 nm.  The peaks also change in relative intensity and broadness.  Qualitatively, it seems that removal of a hydrogen bond causes red shifts in the absorptions, while putative addition of a hydrogen bond causes blue shifts.  Changing the hydrophobic region of the cluster does not make significant changes to the UV-Visible spectrum. These bands are thought to arise from S→Fe ligand to metal charge transfer bands.  The larger magnitude change for G156S is expected since the mutation is designed to make a new hydrogen bond to the S* of the cluster in contrast to changing a hydrogen bond to the S_g of a Cys. 

pH dependent UV-Visible spectra of the wild type and each mutant have also been determined.  The wild type Rieske protein absorption at 458 nm blue shifts to 436 nm and increases in intensity as a function of pH. This change in wavelength is consistent with an increase in the sigma donation ability of the histidines as they are deprotonated.  In a general, qualitative interpretation, changing the sigma donation of two ligands would raise the relative energy of the antibonding e orbitals of the tetrahedral Fe atom(s), resulting in the higher energy absorption needed.

Efforts toward measuring the reduction potential have also been made.  We have made our own pyrolytic graphite electrode, the type of which has been used previously in the literature to measure reduction potential of Rieske proteins.  We have been able to acquire data and are learning how to process the data to compare the measured reduction potential to the values in the literature.  Once we have reproduced the low pH reduction potential reported for the wild type protein, the reduction potential at multiple pH values will be obtained and then repeated for each mutant.