Structural and Functional Characterization of Cyanoglobin

Pennsylvania State Univ University Park, University Park PA

Investigators

Abstract

The hemoglobin superfamily of proteins has representatives in all kingdoms of organisms. In vertebrates, hemoglobin and its close relative myoglobin are responsible for the transport and storage of molecular oxygen. The relationship between three-dimensional structure and function in these two highly specialized proteins has been explored extensively and is well understood. In invertebrates, hemoglobins exhibit a broader range of physiological roles reflecting essential aspects of globin fold evolution, oxygen utilization, and heme reactivity. For these proteins, however, the connection between structure and function is incompletely defined and it is not yet possible to anticipate chemical properties on the basis of the amino acid sequences. This lack of knowledge limits the usefulness of genome sequencing data and the interpretation of evolutionary relationships. The goal of the project is to investigate primitive hemoglobins from the non-nitrogen fixing cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002. Both are hexacoordinate hemoglobins utilizing two histidines to ligate the iron ion, and both undergo a facile post-translational attachment of the heme group apparently unique to them. Biophysical methods (NMR spectroscopy in vitro and in vivo, optical spectroscopy) and biochemical methods (site-directed mutagenesis, genetic manipulations, phenotypic characterization) will be applied to investigate the role of Synechococcus hemoglobin in vivo; determine the three-dimensional structure of Synechocystis and Synechococcus proteins in solution before and after they have undergone the heme modification; and analyze the molecular determinants of heme coordination and reactivity. Comparison will be made to other invertebrate hemoglobins to develop an atomic level understanding of chemical and biochemical properties. The research component of the project provides training opportunities for graduate and undergraduate students and, through a collaboration focused on cyanobacterial physiology and biochemistry, establishes a mechanism for multidisciplinary interactions between the Chemistry department and the Biochemistry and Molecular Biology department. The project also contains three specific educational components that will enhance further its broader impact. The first is the development of three-dimensional visualization modules for an advanced undergraduate biological chemistry course and the inclusion in this course of research-related hemoglobin topics. This is designed to bring the material to life and encourage chemistry students to pursue careers with a life sciences slant. The second is the development of a short course in communication skills aimed at graduate students. The emphasis will be placed on how to convey sophisticated scientific concepts and issues to a general audience. The third is to continue the organization and increase the frequency of inter-departmental structural biology seminars, for which the speakers are advanced students. These activities are expected to prepare undergraduate, graduate, and post-doctoral students for professional careers in academia, industry, and government.