SUMMARY The advent of heme during evolution allowed organisms possessing this compound to safely and efficiently carry out a variety of chemical reactions that otherwise were difficult or impossible. While it was long assumed that a single heme biosynthetic pathway existed in nature, over the past decade, it has become clear that there are three distinct pathways among prokaryotes, although all three pathways utilize a common initial core of three enzymes to produce the intermediate uroporphyrinogen III. The most ancient pathway and the only one found in the Archaea converts siroheme to protoheme via an oxygen-independent four-enzyme-step process. Bacteria utilize the initial core pathway but then add one additional common step to produce coproporphyrinogen III. Following this step, Gram-positive organisms oxidize coproporphyrinogen III to coproporphyrin III, insert iron to make coproheme, and finally decarboxylate coproheme to protoheme, whereas Gram-negative bacteria first decarboxylate coproporphyrinogen III to protoporphyrinogen IX and then oxidize this to protoporphyrin IX prior to metal insertion to make protoheme. In order to adapt to oxygen-deficient conditions, two steps in the bacterial pathways have multiple forms to accommodate oxidative reactions in an anaerobic environment. The regulation of these pathways reflects the diversity of bacterial metabolism. This diversity, along with the late recognition that three pathways exist, has significantly slowed advances in this field such that no single organism's heme synthesis pathway regulation is currently completely characterized.

The bacterial iron response regulator (Irr) protein mediates irondependent regulation of heme biosynthesis. Pulse-chase and immunoprecipitation experiments showed that Irr degraded in response to 6 M iron with a half-life of Ϸ30 min and that this regulated stability was the principal determinant of control by iron. Irr contains a heme regulatory motif (HRM) near its amino terminus. A role for heme in regulation was implicated by the retention of Irr in heme synthesis mutants in the presence of iron. Addition of heme to low iron (0.3 M) cultures was sufficient for the disappearance of Irr in cells of the wild-type and heme mutant strains. Spectral and binding analyses of purified recombinant Irr showed that the protein bound heme with high affinity and caused a blue shift in the absorption spectrum of heme to a shorter wavelength. A Cys 29 3 Ala substitution within the HRM of Irr (IrrC29A) abrogated both high affinity binding to heme and the spectral blue shift. In vivo turnover experiments showed that, unlike wild-type Irr, IrrC29A was stable in the presence of iron. We conclude that iron-dependent degradation of Irr involves direct binding of heme to the protein at the HRM. The findings implicate a regulatory role for heme in protein degradation and provide direct evidence for a functional HRM in a prokaryote.

Heme is a ubiquitous macromolecule that serves as the active group of proteins involved in many cellular processes. The multienzyme pathway for heme formation culminates with the insertion of iron into a protoporphyrin ring. The cytotoxicity of porphyrins suggests the need for coordination of its biosynthesis with iron availability. We isolated a mutant strain of the bacterium Bradyrhizobium japonicum that, under iron limitation, accumulated protoporphyrin and showed aberrantly high expression of hemB, an iron-regulated gene encoding the heme synthesis enzyme ␦-aminolevulinic acid dehydratase. The strain carries a loss of function mutation in irr, a newly described gene that encodes a putative member of the GntR family of bacterial transcriptional regulators. Irr accumulated only under iron limitation, and turned over rapidly upon an increase in iron availability. A separate role for Irr in controlling the cellular iron level was inferred based on a deficiency in high affinity iron transport activity in the irr strain, and suggests that regulation of the heme pathway is coordinated with iron homeostasis. A high level of protoporphyrin accumulation is not a normal consequence of nutritional iron deprivation, thus a mechanism for iron-dependent control of heme biosynthesis may be present in other organisms.

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The heme biosynthetic pathway culminates with the insertion of iron into protoporphyrin catalyzed by ferrochelatase. The Bradyrhizobium japonicum iron response regulator (Irr) protein represses the pathway at an early step under iron limitation to prevent protoporphyrin synthesis from exceeding iron availability. Here, we show that Irr interacts directly with ferrochelatase and responds to iron via the status of heme and protoporphyrin localized at the site of heme synthesis. In the presence of iron, ferrochelatase inactivates Irr, followed by heme-dependent Irr degradation to derepress the pathway. Under iron limitation, protoporphyrin relieves the inhibition of Irr by ferrochelatase, probably by promoting protein dissociation, allowing genetic repression. Thus, metabolic control of the heme pathway involves a regulatory function of a biosynthetic enzyme to affect gene expression. Furthermore, heme can serve as a signaling molecule without accumulating freely in cells.