Succinate dehydrogenase

Mitochondrial and many bacterial SQRs are composed of four structurally different subunits: two hydrophilic and two hydrophobic. The first two subunits, a flavoprotein (SdhA) and an iron-sulfur protein (SdhB), form a hydrophilic head where enzymatic activity of the complex takes place. SdhA contains a covalently attached flavin adenine dinucleotide (FAD) cofactor and the succinate binding site and SdhB contains three iron-sulfur clusters: [2Fe-2S], [4Fe-4S], and [3Fe-4S]. The second two subunits are hydrophobic membrane anchor subunits, SdhC and SdhD. Human mitochondria contain two distinct isoforms of SdhA (Fp subunits type I and type II), these isoforms are also found in Ascaris suum and Caenorhabditis elegans.[3] The subunits form a membrane-bound cytochrome b complex with six transmembrane helices containing one heme b group and a ubiquinone-binding site. Two phospholipid molecules, one cardiolipin and one phosphatidylethanolamine, are also found in the SdhC and SdhD subunits (not shown in the image). They serve to occupy the hydrophobic space below the heme b. These subunits are displayed in the attached image. SdhA is green, SdhB is teal, SdhC is fuchsia, and SdhD is yellow. Around SdhC and SdhD is a phospholipid membrane with the intermembrane space at the top of the image.[4]

Two distinctive ubiquinone binding sites can be recognized on mammalian SDH – matrix-proximal Q_P and matrix-distal Q_D. Ubiquinone binding site Qp, which shows higher affinity to ubiquinone, is located in a gap composed of SdhB, SdhC, and SdhD. Ubiquinone is stabilized by the side chains of His207 of subunit B, Ser27 and Arg31 of subunit C, and Tyr83 of subunit D. The quinone ring is surrounded by Ile28 of subunit C and Pro160 of subunit B. These residues, along with Il209, Trp163, and Trp164 of subunit B, and Ser27 (C atom) of subunit C, form the hydrophobic environment of the quinone-binding pocket Qp.[6] In contrast, ubiquinone binding site Q_D, which lies closer to inter-membrane space, is composed of SdhD only and has lower affinity to ubiquinone.[7]

SdhA provides the binding site for the oxidation of succinate. The side chains Thr254, His354, and Arg399 of subunit A stabilize the molecule while FAD oxidizes and carries the electrons to the first of the iron-sulfur clusters, [2Fe-2S].[8] This can be seen in image 5.

The succinate-binding site and ubiquinone-binding site are connected by a chain of redox centers including FAD and the iron-sulfur clusters. This chain extends over 40 Å through the enzyme monomer. All edge-to-edge distances between the centers are less than the suggested 14 Å limit for physiological electron transfer.[4] This electron transfer is demonstrated in image 8.

SdhE
Solution NMR structure of protein NMA1147 from Neisseria meningitidis. Northeast structural genomics consortium target mr19
Identifiers
Symbol	SdhE
Pfam	PF03937
InterPro	IPR005631
Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

In molecular biology, the protein domain named Sdh5 is also named SdhE which stands for succinate dehydrogenase protein E. In the past, it has also been named YgfY and DUF339.[9] Another name for SdhE is succinate dehydrogenase assembly factor 2 (Sdhaf2).[10] This protein belongs to a group of highly conserved small proteins found in both eukaryotes and prokaryotes, including NMA1147 from Neisseria meningitidis [11] and YgfY from Escherichia coli.[12] The SdhE protein is found on the mitochondrial membrane is it is important for creating energy via a process named the electron transport chain.[9]

The function of SdhE has been described as a flavinator of succinate dehydrogenase. SdhE works as a co-factor chaperone that incorporates FAD into SdhA. This results in SdhA flavinylation which is required for the proper function succinate dehydrogenase. Studies indicate that SdhE is required by bacteria in order to grow on succinate, using succinate as its only source of carbon and additionally for the function, of succinate dehydrogenase, a vital component of the electron transport chain which produces energy.[9]

The structure of these proteins consists of a complex bundle of five alpha-helices, which is composed of an up-down 3-helix bundle plus an orthogonal 2-helix bundle.[12]

SdhE interacts with the catalytic subunit of the succinate dehydrogenase (SDH) complex.[13]

The human gene named SDH5, encodes for the SdhE protein. The gene itself is located in the chromosomal position 11q13.1. Loss-of-function mutations result in paraganglioma, a neuroendocrine tumour.[13]

The recent studies which suggest SdhE is required for bacterial flavinylation contradict previous thoughts on SdhE. It was originally proposed that FAD incorporation into bacterial flavoproteins was an autocatalytic process. Recent studies now argue that SdhE is the first protein to be identified as required for flavinylation in bacteria. Historically, the SdhE protein was once considered a hypothetical protein.[9] YgfY was also thought to be involved in transcriptional regulation.[12]

Little is known about the exact succinate oxidation mechanism. However, the crystal structure shows that FAD, Glu255, Arg286, and His242 of subunit A (not shown) are good candidates for the initial deprotonation step. Thereafter, there are two possible elimination mechanisms: E2 or E1cb. In the E2 elimination, the mechanism is concerted. The basic residue or cofactor deprotonates the alpha carbon, and FAD accepts the hydride from the beta carbon, oxidizing the bound succinate to fumarate—refer to image 6. In E1cb, an enolate intermediate is formed, shown in image 7, before FAD accepts the hydride. Further research is required to determine which elimination mechanism succinate undergoes in Succinate Dehydrogenase. Oxidized fumarate, now loosely bound to the active site, is free to exit the protein.

After the electrons are derived from succinate oxidation via FAD, they tunnel along the [Fe-S] relay until they reach the [3Fe-4S] cluster. These electrons are subsequently transferred to an awaiting ubiquinone molecule within the active site. The Iron-Sulfur electron tunneling system is shown in image 9.

Image 8: Ubiquinone reduction mechanism.

Image 9: Electron carriers of the SQR complex. FADH₂, iron-sulfur centers, heme b, and ubiquinone.

The O1 carbonyl oxygen of ubiquinone is oriented at the active site (image 4) by hydrogen bond interactions with Tyr83 of subunit D. The presence of electrons in the [3Fe-4S] iron sulphur cluster induces the movement of ubiquinone into a second orientation. This facilitates a second hydrogen bond interaction between the O4 carbonyl group of ubiquinone and Ser27 of subunit C. Following the first single electron reduction step, a semiquinone radical species is formed. The second electron arrives from the [3Fe-4S] cluster to provide full reduction of the ubiquinone to ubiquinol. This mechanism of the ubiquinone reduction is shown in image 8.

Although the functionality of the heme in succinate dehydrogenase is still being researched, some studies have asserted that the first electron delivered to ubiquinone via [3Fe-4S] may tunnel back and forth between the heme and the ubiquinone intermediate. In this way, the heme cofactor acts as an electron sink. Its role is to prevent the interaction of the intermediate with molecular oxygen to produce reactive oxygen species (ROS). The heme group, relative to ubiquinone, is shown in image 4.

It has also been proposed that a gating mechanism may be in place to prevent the electrons from tunneling directly to the heme from the [3Fe-4S] cluster. A potential candidate is residue His207, which lies directly between the cluster and the heme. His207 of subunit B is in direct proximity to the [3Fe-4S] cluster, the bound ubiquinone, and the heme; and could modulate electron flow between these redox centers.[16]

To fully reduce the quinone in SQR, two electrons as well as two protons are needed. It has been argued that a water molecule (HOH39) arrives at the active site and is coordinated by His207 of subunit B, Arg31 of subunit C, and Asp82 of subunit D. The semiquinone species is protonated by protons delivered from HOH39, completing the ubiquinone reduction to ubiquinol. His207 and Asp82 most likely facilitate this process. Other studies claim that Tyr83 of subunit D is coordinated to a nearby histidine as well as the O1 carbonyl oxygen of ubiquinone. The histidine residue decreases the pKa of tyrosine, making it more suitable to donate its proton to the reduced ubiquinone intermediate.