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The magnesium-insertion step of chlorophyll biosynthesis is a two-stage reaction.

ABSTRACT: Mg(2+)-chelatase catalyses the first step unique to chlorophyll synthesis, namely the insertion of Mg2+ into protoporphyrin IX. When pea (Pisum sativum L., cv. Spring) chloroplasts are lysed in a buffer lacking Mg2+ and the thylakoids removed by centrifugation, the remaining mixture of light membranes and soluble proteins (LM/S) has high Mg(2+)-chelatase activity. Several lines of evidence are presented to show that the Mg2+ insertion catalysed by this preparation is a two-step reaction consisting of activation followed by Mg2+ chelation. An activated state of Mg(2+)-chelatase is achieved by preincubating LM/S with ATP. The activated state is observed as the elimination of the approx. 6 min lag in the rate of Mg2+ chelation on addition of the porphyrin substrate. The activity of LM/S assayed at low protein concentrations can be greatly enhanced by preincubating at high protein concentrations (12 mg/ml is optimal). This activation effect requires the presence of both LM and S fractions, as well as ATP. Both steps require ATP, but at different concentrations; the first step is optimal at > 0.5 mM (EC50 = 0.3 mM) and the second step is optimal at 0.3 mM (EC50 < 0.2 mM). ATP in the first step could be replaced by ATP[S]; this analogue could not sustain activity in the second step. This activated state was stable for at least 30 min at room temperature, but chilling of preincubated LM/S on ice for 30 min caused an almost complete loss of the activated state.

SUBMITTER: Walker CJ

PROVIDER: S-EPMC1138051 | biostudies-other | 1994 Apr

REPOSITORIES: biostudies-other

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Project description:Magnesium chelatase is a conserved enzyme complex responsible for the first committed step of chlorophyll biosynthesis in photosynthetic organisms, which is the addition of magnesium to the chlorophyll precursor, protoporphyrin IX. The complex is composed of the catalytic subunit ChlH, the bridging subunit ChlD, and the subunit ChlI, which serves as the motor that drives the entire complex. Although the enzyme is well-characterized functionally, high-resolution structures are available only for individual subunits. Hence, the full assembly and molecular mechanism of the enzyme complex remain unknown. Here, we used cryogenic electron microscopy, supported by biochemical analysis and mass photometry, to determine the structures of the ChlI motor subunit of magnesium chelatase under turnover conditions in the presence of ATP. Our data reveal the molecular details of ChlI oligomerization and conformational dynamics upon ATP binding and hydrolysis. These findings provide new insights into the mechanistic function of ChlI and its implications for the entire magnesium chelatase complex machinery. IMPORTANCE Photosynthesis is an essential life process that relies on chlorophyll. In photosynthetic organisms, chlorophyll synthesis involves multiple steps and depends on magnesium chelatase. This enzyme complex is responsible for inserting magnesium into the chlorophyll precursor, but the molecular mechanism of this process is not fully understood. By using cryogenic electron microscopy and conducting functional analyses, we have discovered that the motor subunit ChlI of magnesium chelatase undergoes conformational changes in the presence of ATP. Our findings offer new insights into how energy is transferred from ChlI to the other components of magnesium chelatase. This information significantly contributes to our understanding of the initial step in chlorophyll biosynthesis and lays the foundation for future studies on the entire process of chlorophyll production.

Project description:The dimeric ?-diketiminato magnesium hydride, [(BDI)MgH]2, reacts at 80 °C with the terminal alkenes, 1-hexene, 1-octene, 3-phenyl-1-propene and 3,3-dimethyl-butene to provide the respective n-hexyl, n-octyl, 3-phenylpropyl and 3,3-dimethyl-butyl magnesium organometallics. The facility for and the regiodiscrimination of these reactions are profoundly affected by the steric demands of the alkene reagent. Reactions with the phenyl-substituted alkenes, styrene and 1,1-diphenylethene, require a more elevated temperature of 100 °C with styrene providing a mixture of the 2-phenylethyl and 1-phenylethyl products over 7 days. Although the reaction with 1,1-diphenylethene yields the magnesium 1,1-diphenylethyl derivative as the sole reaction product, only 64% conversion was achieved over a 21 day timeframe. Reactions with the ?,?-dienes, 1,5-hexadiene and 1,7-octadiene, provided divergent results. The initial 5-alkenyl magnesium reaction product of the shorter chain diene undergoes 5-exo-trig cyclisation via intramolecular carbomagnesiation to provide a cyclopentylmethyl derivative, which was shown by X-ray diffraction analysis to exist as a three-coordinate monomer. In contrast, 1,7-octadiene provided a mixture of two compounds, a magnesium oct-7-en-1-yl derivative and a dimagnesium-octane-1,4-diide, as a result of single or two-fold activation of the terminal C[double bond, length as m-dash]C double bonds. The magnesium hydride was unreactive towards internal alkenes apart from the strained bicycle, norbornene, allowing the characterisation of the resultant three-coordinate magnesium norbornyl derivative by X-ray diffraction analysis. Computational analysis of the reaction between [(BDI)MgH]2 and 1-hexene using density functional theory (DFT) indicated that the initial Mg-H/C[double bond, length as m-dash]C insertion process is rate determining and takes place at the intact magnesium hydride dimer. This exothermic reaction (?H = -14.1 kcal mol-1) traverses a barrier of 18.9 kcal mol-1 and results in the rupture of the dinuclear structure into magnesium alkyl and hydride species. Although the latter three-coordinate hydride derivative may be prone to redimerisation, it can also provide a further pathway to magnesium alkyl species through its direct reaction with a further equivalent of 1-hexene, which occurs via a lower barrier of 15.1 kcal mol-1. This Mg-H/C[double bond, length as m-dash]C insertion reactivity provides the basis for the catalytic hydrosilylation of terminal alkenes with PhSiH3, which proceeds with a preference for the formation of the anti-Markovnikov organosilane product. Further DFT calculations reveal that the catalytic reaction is predicated on a sequence of Mg-H/C[double bond, length as m-dash]C insertion and classical Si-H/Mg-C ?-bond metathesis reactions, the latter of which, with a barrier height of 24.9 kcal mol-1, is found to be rate determining.

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The magnesium-insertion step of chlorophyll biosynthesis is a two-stage reaction.

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