Date of Award

1997

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Alan J. Biel

Abstract

Rhodobacter capsulatus, a purple non-sulfur photosynthetic bacterium, synthesizes four tetrapyrrole end products: heme, bacteriochlorophyll, siroheme and vitamin B$\sb{12}$ via the common tetrapyrrole biosynthetic pathway. The biosynthesis of tetrapyrroles in R. capsulatus is a complex and precisely controlled process which has been under extensive investigation. In order to study the regulation of tetrapyrrole biosynthesis, the R. capsulatus hemH gene was cloned by complementation of an E. coli $\Delta hemH$ mutant and sequenced. The enzyme ferrochelatase, encoded by the hemH gene, catalyzes the insertion of ferrous iron into protoporphyrin IX, thus producing heme. The consensus ferrochelatase signature sequence was found in the R. capsulatus hemH gene sequence, with the exception of one amino acid substitution. Mapping of the hemH gene to the R. capsulatus chromosome confirmed that the hem genes located so far are not arranged in a cluster. Overexpression of the hemH gene in R. capsulatus was used to study the effect of excess heme on the regulation of tetrapyrrole biosynthesis. The overexpression resulted in increased ferrochelatase activity, decreased aminolevulinate synthase activity and reduced levels of porphyrins and bacteriochlorophyll. This was the first evidence for feedback inhibition of aminolevulinate synthase by heme in R. capsulatus. These in vivo results were also confirmed by in vitro experiments. In order to determine if oxygen and heme participate in a single regulatory mechanism, a coproporphyrin-accumulating R. capsulatus mutant was grown in the presence of execs heme under high and low oxygen tension. The results clearly indicate that regulation of tetrapyrrole biosynthesis by heme is separate from regulation by oxygen.

ISBN

9780591591415

Pages

104

Share

COinS