Complex Formation of Protoporphyrinogen IX Oxidase and Ferrochelatase during the Tetrapyrrole Biosynthesis in Thermosynechococcus elongatus

Coproporphyrinogen oxidase (CPO), protoporphyrinogen oxidase (PPO) and ferrochelatase (FC) catalyze the three terminal steps of the heme biosynthetic pathway to form protoheme IX. For the protection of the highly toxic intermediate protoporphyrin IX, a protein complex between CPO, PPO and FC was proposed based on the solved crystal structure of tobacco PPO (Koch et al., 2004). It was objective in this thesis to proof the existance and determine the localization of the proposed complex in T. elongatus cells. Co-immunoprecipitation experiments and double-immunogold labelling in combination with electron microscopy were employed. First recombinant T. elongatus HemF (CPO), HemY (PPO) and HemH (FC) were produced and chromatographically purified subsequently, polyclonal rabbit antibodies were raised against the three enzymes. Complex formation of PPO with FC was demonstrated for T. elongatus cell free extracts via co-immunoprecipitation for the first time. These in vitro findings were further confirmed using electron microscopy and double-immunogold labelled PPO and FC. PPO - FC complexes were detected in T. elongatus thylakoid membranes while CPO was not involved in complex formation. Obtained data lay the basis for the in-depth analysis of the cellular arrangement of tetrapyrrole biosynthetic enzymes. The fidelity of protein biosynthesis is dependent on the reliable charging of each tRNA with its cognate amino acid. However, many bacteria lack a glutaminyl-tRNA synthetase. In these organisms tRNAGln is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase (GluRS). The resulting misacylated Glu-tRNAGln is then converted to Gln-tRNAGln by a tRNA-dependent amidotransferase through the amide addition to the tRNA-bound glutamate. This thesis provided experimental proof that Thermosynechococcus elongatus GluRS is indeed a non-discriminating tRNA synthetase. For this purpose the kinetic parameters for the synthesis of Glu-tRNAGlu and Glu-tRNAGln were determined. Therefore T. elongatus tRNAGlu and tRNAGln were purified. It was shown that T. elongatus GluRS accommodates and charges both tRNAGlu as well as tRNAGln. Obtained results were discussed in the light of solved T. elongatus GluRS crystal structure.