Crystal structure of the nitrogenase-like dark operative protochlorophyllide oxidoreductase catalytic complex (ChlN/ChlB)2

Abstract

During (bacterio)chlorophyll biosynthesis of many photosynthetically active organisms, dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the two-electron reduction of ring D of protochlorophyllide to form chlorophyllide. DPOR is composed of the subunits ChlL, ChlN, and ChlB. Homodimeric ChlL(2) bearing an intersubunit [4Fe-4S] cluster is an ATP-dependent reductase transferring single electrons to the heterotetrameric (ChlN/ChlB)(2) complex. The latter contains two intersubunit [4Fe-4S] clusters and two protochlorophyllide binding sites, respectively. Here we present the crystal structure of the catalytic (ChlN/ChlB)(2) complex of DPOR from the cyanobacterium Thermosynechococcus elongatus at a resolution of 2.4 A. Subunits ChlN and ChlB exhibit a related architecture of three subdomains each built around a central, parallel beta-sheet surrounded by alpha-helices. The (ChlN/ChlB)(2) crystal structure reveals a [4Fe-4S] cluster coordinated by an aspartate oxygen alongside three cysteine ligands. Two equivalent substrate binding sites enriched in aromatic residues for protochlorophyllide substrate binding are located at the interface of each ChlN/ChlB half-tetramer. The complete octameric (ChlN/ChlB)(2)(ChlL(2))(2) complex of DPOR was modeled based on the crystal structure and earlier functional studies. The electron transfer pathway via the various redox centers of DPOR to the substrate is proposed.

FIGURE 1.

Catalytic reaction of the DPOR and schematic representation of the subunit architecture. A, ATP-dependent DPOR catalysis involves the two-electron reduction of the C17=C18 double bond of Pchlide to form Chlide. R is either an ethyl or a vinyl residue. B, two ChlL₂ dimers interact with the heterotetrameric (ChlN/ChlB)₂ complex to form hetero-octameric DPOR during catalysis. The four intersubunit [4Fe-4S] clusters are marked as red squares.

FIGURE 2.

Structure of the (ChlN/ChlB)₂ heterotetramer of DPOR. A ribbon diagram of the (ChlN/ChlB)₂ structure in two mutually perpendicular views, with subunits ChlN and ChlB, respectively, depicted in green and orange is shown. Note that the asymmetric unit contains only one ChlN/ChlB half-tetramer, the heterotetramer involving a crystallographic two-fold rotation axis indicated by appropriate spindle and dashed line. The total accessible surface area between ChlN and ChlB in the heterodimer is 2485 Ų, whereas those between ChlB/ChlB′ and ChlN/ChlB′ are 1560 and 1074 Ų. ChlN and ChlN′ do not share a common interface. The [4Fe-4S] centers are indicated by clusters of red spheres.

FIGURE 3.

Structure-based sequence alignment of ChlN/ChlB. The structure-based amino acid sequence alignment of ChlN and ChlB results in 366 aligned residues and a sequence identity of 13.7%. α-Helices and β-strands are marked as α1–α20 and β1–β12, and the three subdomains are marked by progressively darker shades of green. Ligands coordinating the [4Fe-4S] cluster (Cys²², Cys⁴⁷, and Cys¹⁰⁷ of ChlN and Asp³⁶ of ChlB) are indicated by red shading. Cys⁹⁵ of ChlB involved in stabilizing the cluster is marked in black. Residues proposed to be involved in substrate and ChlL₂ binding are indicated by cyan and magenta boxes.

FIGURE 4.

Comparison of subdomains of ChlN and ChlB. ChlN (shades of green) and ChlB (yellow to orange) each consist of three similar subdomains. Each subunit bears a central, parallel β-sheet surrounded by α-helices. The first subdomain of ChlN and ChlB serves to coordinate the [4Fe-4S] and is involved in ChlL₂ binding. The second subdomain appears to have a largely structural role in positioning the remaining two domains but may also be involved in substrate recognition. The third subdomain of ChlN and ChlB is involved in forming the active site channel and in substrate recognition.

FIGURE 5.

Coordination of the [4Fe-4S] cluster. The crystal structure reveals one intersubunit [4Fe-4S] cluster per heterodimer in the asymmetric unit or two [4Fe-4S] clusters per (ChlN/ChlB)₂ complex. Residues coordinating the cluster are 3 cysteine residues (Cys²², Cys⁴⁷, Cys¹⁰⁷) of subunit ChlN and a unique aspartate residue (Asp³⁶) of subunit ChlB. Coordinating distances are indicated in Angstroms.

FIGURE 6.

Cut-away of the proposed binding site of DPOR. The surface representation of the (ChlN/ChlB)₂ complex was cut away to reveal one of the two equivalent proposed substrate binding sites and its spatial positioning relative to the iron-sulfur cluster. A substrate molecule has been included for purely qualitative purposes; only a co-crystal structure would provide all details of interaction with a high degree of reliability. Conserved residues are indicated by shades of orange (dark orange, conserved; white, not conserved). Note that most residues in the immediate vicinity of the cluster and of the proposed binding site are highly conserved. The active site entrance is created by residues from ChlN as well as both symmetry-related ChlB subunits. The approximate position of the MoFe cofactor in the related nitrogenase structure is indicated by a dashed circle.

FIGURE 7.

Model of the complete hetero-octameric DPOR and distance between cofactors. Left, a qualitative, theoretical model of the complete DPOR was created by combining the heterotetrameric (ChlN/ChlB)₂ core complex from T. elongatus (this study) and ChlL₂ from R. sphaeroides (PDB code 3FWY) (16), based on the crystal structure of hetero-octameric nitrogenase (PDB code 1G21) (54). ChlN (ChlN′) and ChlB (ChlB′) are shown in shades of green (partly transparent), ChlL₂ is shown in shades of blue. Note the good surface complementarity between the subcomplexes. Right, the cofactors and the substrate of DPOR derived from the models on the left and Fig. 5 are shown in stick or ball-and-stick representation. Edge-to-edge distances are indicated in Angstroms, and distances from theoretical models are indicated by asterisks.