Flavodoxin (PDB ID = 1RCF)
Created by Melissa Belardi
Flavodoxin in the long chain, oxidized form present in Anabaena 7120 is a small flavoprotein that aids in electron transfer reactions. It has a molecular weight of 18832.62 (g/mol) and an isoelectric point of 4.17. This protein is referred to as a flavoprotein because it contains a flavin mononucleotide (FMN) that serves as a cofactor in an electron transfer reaction (1). The organism that this flavoprotein is found in, Anabaena, is a cyanobacteria, which is commonly known as blue-green algae. Since blue-green algae is a form of plankton residing on the surface of a body of water, it undergoes photosynthesis to acquire energy from the sun. A very important component of photosynthesis is the absorption of light by photosystems allowing for the transfer of electrons by electron carriers. Iron, in the case of cyanobacteria, is important for the organism because it works to carry electrons from the photosystem in the basteria's cytoplasmic membrane to ferredoxin reductase. When sufficient iron is not available in Anabaena to produce ferredoxin, the protein flavodoxin works in its place to carry electrons from Photosystem I to the enzyme, ferredoxin reductase. As an electron carrier, flavodoxin fluctuates between three different redox states: fully reduced, partially reduced, and oxidized. This allows it to pick up electrons and carry them between Photosystem I and ferredoxin reductase, ultimately allowing for the reduction of NADPH to NADP+ (2).
The flavodoxin in Anabaena is 169 residues long. When comparing this primary structure to the sequence of other proteins in the 'protein blast' databank, the flavodoxin from the bacteria Anacystis nidulans has a significantly similar sequence (5). Both strains of bacteria perform the same redox reaction to obtain energy from the sun. Since the protein from Anacystis nidulans is similar in sequence with 169 amino acids and a low E value of 2e-65 (or 2 x10^-65), the primary structure of the flavodoxin may be genetically conservedin blue-green algae. The low E value indicates similar primary structure. Usingthe Dali server to search for proteins with similar tertiary structure, aspecific domain of Escherichia coli sulfite reductase showed similarity to this Anabaena’s flavodoxin. Only a specific part or domain of the sulfite reductase in E.Coli has a similar structure, not the entire enzyme itself. The function of this sequence is to aid in the reduction of NADPH much like that of the flavodoxin in the plankton described above. However, in this case it aids a different enzyme in reducing NADPH to NADP+(4). This electron transfer is used to convert sulfiteinto sulfide as opposed to the photosynthetic function used in the Anabaena. The Z score for these two proteins is 18.6, indicating that the tertiary structures are significantly similar as a Z score greater than 2 is significant. The root mean deviation squared is given as 2.1 indicating that the superimposition of the backbones of the proteins varies by 2.1 angstroms. Although this is not an ideal score, it islow enough to consider the structures similar.
The structure of the trigonal form of recombinant oxidized flavodoxin from Anabaena 7120 is a monomer that contains a central five-stranded parallel betasheet surrounded by five helices (7). The beta-sheet is sandwiched by a pair ofhelices on either side. Residues 4-9, 30-36, 48-54, 81-89 and 115-117/141-143 are involved with the strands of the beta sheet (2). In the fifth strand of thebeta sheet, there exists a long loop consisting of 14 amino acids between residues 117-141 (2). The fifth strand alsocontains a short anti-parallel sheet that interacts by hydrogen bonding between Trp-120 and Phe- 138 3-10-helix (2) indicating that there are three residues and ten atoms per turn of the helix (8). Although four out of the five helices can be described as alpha helices, the alpha-2 helix can also be described as a 3/10 helix. It is suggested that this irregularity is due to an interaction between residues 70-76 of the alpha-2 helix and Arg-112 (2). In summary, the secondary structure consists of a beta sheet with a pair of helices on either side and an extra 3/10 helix. The residues that are involved in the four alpha helices and the 3/10 helix are as follows: 12-26, 70-76,100-110,151-168, and 40-46 respectively (2). There are three different conformations the protein can adopt when it is bound to its ligand, a flavin mononucleotide, on its 'southern edge'. When this form of flavodoxin is bound to FMN, it transfers electrons from photosystem I to ferredoxin-reductase (1). During this process, it fluctuates between its semi-reduced form with a semiquinone presence, and a fully reduced state. If the flavin mononucleotide is semiquinone, then one electron has been gained, and if it is hydroquinone then two electrons have been gained by the mononucleotide (2). When the protein is in contact with air, it becomes fully oxidized. The ligand makes the protein more likely to be reduced as it is a cofactor in the electron transfer process(1).
A single flavin mononucleotide (FMN) is bound to the C-terminus of the beta sheet as a ligand to the apoprotein. Its dimethyl benzene edge is exposed to solvent (2). Since this particular flavodoxin is a long-chain flavodoxin with 169 residues (7), it contains a lobe of about 20 residues inserted into the middle of the Beta-5 strand (2). This lobe has no direct interaction with the flavin mononucleotide. However, the FMN molecule has an interaction with residues in loops between thebeta strands and with N-terminal residues of the alpha-1 helix. The FMN is tightly bound to the protein by a number of interactions. The following residues interact with FMN and each other to form a binding pocket: Thr-88, Pro-55, Thr-12, Trp-57, Gly-89, Asn-58, Asp-146, Asp-90, and Tyr-94. Many of these interactions occur between the phosphate oxygen atoms of the FMN at the N-terminal of the helix and with residues in the loop that occurs before the helix (7). These interactions correctly orient the helix dipole to allow binding of phosphate to the N-terminal of the alpha-1 helix (2). In order to stabilize the binding loop, an oxygen in the hydroxyl group of Thr-10 is hydrogen bonded to the nitrogen of Gly-13. The phosphate binding loop also makes a hydrogen bond by the interaction of the nitrogen in Trp-57 ring and the hydrogen of the second oxygen from the phosphophate group. This forms one of the faces that encloses the flavin as it binds. In addition, the alloxazine ring of the FMN interacts directly with the backbone atoms of the protein on the loop of amino acids 58-62 between the third beta strand and second alpha helix, which occurs in the reverse direction. The residues 90-99 in the loop between the fourth beta strand and the third alpha helix also interact with the alloxazine ring from the FMN. Within the ring, N-1 and O-2 interact with a hydrogen of the amide in Asp-90 forming a hydrogen bond with both atoms simultaneously. The amide nitrogen of Gln-99 interacts with the O-2 in the alloxazine ring by hydrogen bond, and finally the carbonyl oxygen of Asn-97 hydrogen bonds with the hydrogen on N-3 in the alloxazine ring (2). When flavin is reduced, one site of protonation is N-5 in the alloxazine ring. Before protonation of FMN, N-5 of alloxazine ring hydrogen bonds with the amide hydrogen of Ile-59. Although there is no atom in the protein that is within a preferred value of 3.2 angstroms from this nitrogen, the backbone nitrogen of Ile-59 is at a distance of 3.6angstroms (2). This region is not solvent accessible and allows this hydrogen bond to occur over a less than ideal distance. The ring in FMN (that contains the nitrogen that is reduced) is surrounded on either side by the two aromatic side chains of residue Tyr-94 and Trp-57 (2). The ring of Tyr-94 is nearly paralell to the FMN ring providing However, this is not the case with Trp-57. The backbone of Trp-57 interacts with a portion of the ring that mimics a pyrimidine, which is relatively polar. The aromatic portion of theTrp-57 side chain interacts with the hydrophobic portion of the flavin ring. The coupling of these interactions causes the ring structures of Trp-57 and FMN to not stack in a parallel fashion.