Chlorophyllide

Magnesium is inserted into protoporphyrin IX

Chlorophyll is characterised by having a magnesium ion coordinated within a ligand called a chlorin. The metal is inserted into protoporphyrin IX by the enzyme magnesium chelatase[1] which catalyzes the reaction EC 6.6.1.1

protoporphyrin IX + Mg²⁺
+ ATP + H
₂O ${\displaystyle \rightleftharpoons }$ ADP + phosphate + Mg-protoporphyrin IX + 2 H⁺

The next step towards the chlorophyllides is the formation of a methyl (CH₃) ester on one of the propionate groups, which is catalysed by the enzyme Magnesium protoporphyrin IX methyltransferase[6] in the methylation reaction EC 2.1.1.11

Mg-protoporphyrin IX + S-adenosylmethionine ${\displaystyle \rightleftharpoons }$ Mg-protoporphyrin IX 13-methyl ester + S-adenosyl-L-homocysteine

The chlorin ring system forms as the esterified propionate sidechain is cyclised on to the main porphyrin ring to form divinylprotochlorophyllide

The chlorin ring system features a five-membered carbon ring E is created when one of the propionate groups of the porphyrin is cyclised to the carbon atom linking the original pyrrole rings C and D. A series of chemical steps catalysed by the enzyme Magnesium-protoporphyrin IX monomethyl ester (oxidative) cyclase[7] gives the overall reaction EC 1.14.13.81

Mg-protoporphyrin IX 13-monomethyl ester + 3 NADPH + 3 H⁺ + 3 O₂ ${\displaystyle \rightleftharpoons }$ divinylprotochlorophyllide + 3 NADP⁺ + 5 H₂O

In barley the electrons are provided by reduced ferredoxin, which can obtain them from photosystem I or, in the dark, from Ferredoxin—NADP(+) reductase: the cyclase protein is named XanL and is encoded by the Xantha-l gene.[8] In anaerobic organisms such as Rhodobacter sphaeroides the same overall transformation occurs but the oxygen incorporated into magnesium-protoporphyrin IX 13-monomethyl ester comes from water in the reaction EC 1.21.98.3.[9]

Two further transformations are required to produce chlorophyllide a. Both are reduction reactions: one converts a vinyl group to an ethyl group and the second adds two atoms of hydrogen to the pyrrole ring D, although the overall aromaticity of the macrocycle is retained. These reactions proceed independently and in some organisms the sequence is reversed.[1] The enzyme divinyl chlorophyllide a 8-vinyl-reductase[10] converts 3,8-divinylprotochlorophyllide to protochlorophyllide in reaction EC 1.3.1.75

3,8-divinylprotochlorophyllide + NADPH + H⁺ ${\displaystyle \rightleftharpoons }$ protochlorophyllide + NADP⁺

The reduction of ring D of protochlorophyllide completes the biosynthesis of chlorophyllide a

This is followed by the reaction EC 1.3.1.33 in which the pyrrole ring D is reduced by the enzyme protochlorophyllide reductase[11]

protochlorophyllide + NADPH + H⁺ ${\displaystyle \rightleftharpoons }$ chlorophyllide a + NADP⁺

This reaction is light-dependent but there is an alternative enzyme, Ferredoxin:protochlorophyllide reductase (ATP-dependent),[12] that uses reduced ferredoxin as its cofactor and is not dependent on light; it performs the a similar reaction EC 1.3.7.7 but with the alternative substrate 3,8-divinylprotochlorophyllide

3,8-divinylprotochlorophyllide + reduced ferredoxin + 2 ATP + 2 H₂O ${\displaystyle \rightleftharpoons }$ 3,8-divinylchlorophyllide a + oxidized ferredoxin + 2 ADP + 2 phosphate

In the organisms which use this alternative sequence of reduction steps, the process is completed by the reaction EC 1.3.7.13 catalysed by an enzyme which can take a variety of substrates and perform the required vinyl-group reduction, for example in this case

3,8-divinylchlorophyllide a + 2 reduced ferredoxin + 2 H⁺ ${\displaystyle \rightleftharpoons }$ chlorophyllide a + 2 oxidized ferredoxin

Chlorophyllide a oxygenase is the enzyme that converts chlorophyllide a to chlorophyllide b[13] by catalysing the overall reaction EC 1.3.7.13

chlorophyllide a + 2 O₂ + 2 NADPH + 2 H⁺ ${\displaystyle \rightleftharpoons }$ chlorophyllide b + 3 H₂O + 2 NADP⁺

Bacteriochlorophyllide a (R=H). Earlier intermediates have a vinyl group or 1-hydroxyethyl group in place of the acetyl group shown.

Bacteriochlorophyll a is a typical example; its biosynthesis has been studied in Rhodobacter capsulatus and Rhodobacter sphaeroides. The first step is the reduction (with trans stereochemistry) of the pyrrole ring B, giving the characteristic 18-electron aromatic system of many bacteriochlorophylls. This is carried out by the enzyme chlorophyllide a reductase, which catalyses the reaction EC 1.3.7.15.

chlorophyllide a + 2 reduced ferredoxin + ATP + H₂O + 2 H⁺ ${\displaystyle \rightleftharpoons }$ 3-deacetyl 3-vinylbacteriochlorophyllide a + 2 oxidized ferredoxin + ADP + phosphate

The next two steps convert the vinyl group first into a 1-hydroxyethyl group and then into the acetyl group of bacteriochlorophyllide a. The reactions are catalysed by chlorophyllide a 3¹-hydratase (EC 4.2.1.165) and bacteriochlorophyllide a dehydrogenase (EC 1.1.1.396) as follows:[2][17]

3-deacetyl 3-vinylbacteriochlorophyllide a + H₂O ${\displaystyle \rightleftharpoons }$ 3-deacetyl 3-(1-hydroxyethyl)bacteriochlorophyllide a

3-deacetyl 3-(1-hydroxyethyl)bacteriochlorophyllide a + NAD⁺ ${\displaystyle \rightleftharpoons }$ bacteriochlorophyllide a + NADH + H⁺

These three enzyme-catalysed reactions can occur in different sequences to produce bacteriochlorophyllide a ready for esterification to the final pigments for photosynthesis. The phytyl ester of bacteriochlorophyll a is not attached directly: rather, the initial intermediate is the ester with R=geranylgeranyl (from geranylgeranyl pyrophosphate) which is then subject to additional steps as three of the sidechain's alkene bonds are reduced.[17]