Single Molecule and Hole-Burning Spectroscopies for the Determination of Molecular Electric Fields in Proteins

Abstract

Research in recent years has been pointing to molecular electric fields, generated by the charge distribution of nuclei and electrons inside molecules, as significant contributor to protein function in a variety of phenomena ranging from enzymatic activity [1,2] to photosynthesis [3]. Gaining detailed, quantitative information about such fields from experiments, however, has remained a difficult task. In this work, we will outline our experimental approach to quantitatively determine the molecular electric fields at the oxygen binding site in the heme proteins myoglobin and hemoglobin using single molecule and hole-burning spectroscopies in combination with quantum-mechanical models for data analysis. While hole-burning measurements average over a subset of proteins in a sample, single protein studies will reveal the distribution of internal molecular electric fields. We will discuss in the framework of our models hole-burning measurements in myoglobin and the dependence of the resulting molecular electric fields in the protein on the input parameters of these models. Moreover, we will present measurements of absorption cross-sections and fluorescence quantum yields of various fluorescent heme derivatives, which will serve as molecular probes of the internal field at the protein active sites. The results on room temperature imaging of single molecules of protoporphyrin IX embedded in thin polymer films demonstrate that such experiments are indeed feasible on the level of single molecules.[1] E.D. Getzoff, D.E. Cabelli, C.L. Fisher, H.E. Parge, M.S. Viezzoli, L. Banci, and R.A. Hallewell, Nature 358, (1992) 347.[2] J.P. Hosler, J.P. Shapleigh, D.M. Mitchell, Y. Kim, M.A. Pressler, C. Georgiou, G.T. Babcock, J.O. Alben, S. Ferguson-Miller, and R.B. Gennis, Biochemistry 35, (1996) 10776.[3] A.P. de Silva and T E. Rice, Chem. Commun., (1999) 163.