A bchD (Magnesium Chelatase) Mutant of Rhodobacter sphaeroides Synthesizes Zinc Bacteriochlorophyll through Novel Zinc-containing Intermediates

Paul R Jaschke,Amelia Hardjasa,Elizabeth L Digby,C Neil Hunter,J Thomas Beatty

doi:10.1074/jbc.m110.212605

Abstract

Heme and bacteriochlorophyll a (BChl) biosyntheses share the same pathway to protoporphyrin IX, which then branches as follows. Fe(2+) chelation into the macrocycle by ferrochelatase results in heme formation, and Mg(2+) addition by Mg-chelatase commits the porphyrin to BChl synthesis. It was recently discovered that a bchD (Mg-chelatase) mutant of Rhodobacter sphaeroides produces an alternative BChl in which Mg(2+) is substituted by Zn(2+). Zn-BChl has been found in only one other organism before, the acidophilic Acidiphilium rubrum. Our objectives in this work on the bchD mutant were to 1) elucidate the Zn-BChl biosynthetic pathway in this organism and 2) understand causes for the low amounts of Zn-BChl produced. The bchD mutant was found to contain a Zn-protoporphyrin IX pool, analogous to the Mg-protoporphyrin IX pool found in the wild type strain. Inhibition of ferrochelatase with N-methylprotoporphyrin IX caused Zn-protoporphyrin IX and Zn-BChl levels to decline by 80-90% in the bchD mutant, whereas in the wild type strain, Mg-protoporphyrin IX and Mg-BChl levels increased by 170-240%. Two early metabolites of the Zn-BChl pathway were isolated from the bchD mutant and identified as Zn-protoporphyrin IX monomethyl ester and divinyl-Zn-protochlorophyllide. Our data support a model in which ferrochelatase synthesizes Zn-protoporphyrin IX, and this metabolite is acted on by enzymes of the BChl pathway to produce Zn-BChl. Finally, the low amounts of Zn-BChl in the bchD mutant may be due, at least in part, to a bottleneck upstream of the step where divinyl-Zn-protochlorophyllide is converted to monovinyl-Zn-protochlorophyllide.

Highlights

Of membrane-bound light-harvesting antenna and reaction center complexes is repressed by high concentrations of oxygen and induced in response to low concentrations of oxygen [1]
To determine which metabolite is present in the bchD mutant, metalloporphyrins extracted from wild type and bchD cells were analyzed by absorption and fluorescence spectroscopy, relative to authentic Mg- and zinc protoporphyrin IX (Zn-PPIX) standards
Because of the different spectral signature of the bchD mutant extract compared with the wild type strain and authentic Mg-PPIX and the similarity to authentic Zn-PPIX, it appeared that the bchD mutant contained Zn-PPIX instead of Mg-PPIX

Summary

Introduction

Of membrane-bound light-harvesting antenna and reaction center complexes (the photosystem) is repressed by high concentrations of oxygen and induced in response to low concentrations of oxygen [1]. PPIX lies at a major branch point: if PPIX is used as a substrate by ferrochelatase (HemH) to insert Fe2ϩ, heme is produced, whereas if PPIX is acted on by Mg-chelatase (BchHID) to insert Mg2ϩ, it is the first step in a long pathway leading to BChl. In R. sphaeroides, ferrochelatase is a monomer and binds to the inner face of the cytoplasmic membrane, there is a wide diversity of subunit composition and localization in different organisms [14]. Transcript levels of the ferrochelatase gene appear to be relatively constant with changes in growth conditions, a shift from aerobic respiratory to anaerobic photosynthetic growth resulted in a modest 2-fold increase in PPIX, protoporphyrin IX; MME, monomethyl ester; NMPP, N-methylprotoporphyrin IX. After the formation of Mg-PPIX, eight enzymatic steps subsequently lead to the creation of BChl (reviewed in Refs. 22 and 23)

Objectives

Results

Conclusion