The Long and Short Flavodoxins

Flavodoxins are well known one-domain α/β electron-transfer proteins that, according to the presence or absence of a ∼20-residue loop splitting the fifth β-strand of the central β-sheet, have been classified in two groups: long and short-chain flavodoxins, respectively. Although the flavodoxins have been extensively used as models to study electron transfer, ligand binding, protein stability and folding issues, the role of the loop has not been investigated. We have constructed two shortened versions of the long-chain Anabaena flavodoxin in which the split β-strand has been spliced to remove the original loop. The two variants have been carefully analyzed using various spectroscopic and hydrodynamic criteria, and one of them is clearly well folded, indicating that the long loop is a peripheral element of the structure of long flavodoxins. However, the removal of the loop (which is not in contact with the cofactor in the native structure) markedly decreases the affinity of the apoflavodoxin-FMN complex. This seems related to the fact that, in long flavodoxins, the adjacent tyrosine-bearing FMN binding loop (which is longer and thus more flexible than in short flavodoxins) is stabilized in its competent conformation by interactions with the excised loop. The modest role played by the long loop of long flavodoxins in the structure of these proteins (and in its conformational stability, see López-Llano, J., Maldonado, S., Jain, S., Lostao, A., Godoy-Ruiz, R., Sanchez-Ruiz, Cortijo, M., Fernández-Recio, J., and Sancho, J. (2004) J. Biol. Chem. 279, 47184–47191) opens the possibility that its conservation in so many species is related to a functional role yet to be discovered. In this respect, we discuss the possibility that the long loop is involved in the recognition of some flavodoxin partners. In addition, we report on a structural feature of flavodoxins that could indicate that the short flavodoxins derive from the long ones. Flavodoxins are well known one-domain α/β electron-transfer proteins that, according to the presence or absence of a ∼20-residue loop splitting the fifth β-strand of the central β-sheet, have been classified in two groups: long and short-chain flavodoxins, respectively. Although the flavodoxins have been extensively used as models to study electron transfer, ligand binding, protein stability and folding issues, the role of the loop has not been investigated. We have constructed two shortened versions of the long-chain Anabaena flavodoxin in which the split β-strand has been spliced to remove the original loop. The two variants have been carefully analyzed using various spectroscopic and hydrodynamic criteria, and one of them is clearly well folded, indicating that the long loop is a peripheral element of the structure of long flavodoxins. However, the removal of the loop (which is not in contact with the cofactor in the native structure) markedly decreases the affinity of the apoflavodoxin-FMN complex. This seems related to the fact that, in long flavodoxins, the adjacent tyrosine-bearing FMN binding loop (which is longer and thus more flexible than in short flavodoxins) is stabilized in its competent conformation by interactions with the excised loop. The modest role played by the long loop of long flavodoxins in the structure of these proteins (and in its conformational stability, see López-Llano, J., Maldonado, S., Jain, S., Lostao, A., Godoy-Ruiz, R., Sanchez-Ruiz, Cortijo, M., Fernández-Recio, J., and Sancho, J. (2004) J. Biol. Chem. 279, 47184–47191) opens the possibility that its conservation in so many species is related to a functional role yet to be discovered. In this respect, we discuss the possibility that the long loop is involved in the recognition of some flavodoxin partners. In addition, we report on a structural feature of flavodoxins that could indicate that the short flavodoxins derive from the long ones. The flavodoxins are electron transfer proteins involved in both photosynthetic and non-photosynthetic reactions, which carry a molecule of non-covalently bound FMN as their only redox center (1Mayhew S.G. Tollin G. Müller F. Chemistry and Biochemistry of Flavoenzymes. III. CRC Press, Inc., Boca Raton, FL1992: 389-426Google Scholar, 2Ludwig M.L. Luschinsky C.L. Müller F. Chemistry and Biochemistry of flavoenzymes. III. CRC Press, Inc., Boca Raton, FL1992: 427-466Google Scholar). Soon after their discovery, it was realized that they could be isolated in two sizes and were accordingly divided in two classes: 1) the short-chain flavodoxins (i.e. Clostridium beijerincki and Desulfovibrio vulgaris flavodoxins) and 2) the long-chain ones (i.e. Synechococcus sp. (strain PCC 7942) and Anabaena sp. (strain PCC 7119) flavodoxins). Once the x-ray structures of representatives of the two groups became available (3Ludwig M.L. Andersen R.D. Mayhew S.G. Massey V. J. Biol. Chem. 1969; 244: 6047-6048Abstract Full Text PDF PubMed Google Scholar, 4Watenpaugh K.D. Sieker L.C. Jensen L.H. Legall J. Dubourdieu M. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 3185-3188Crossref PubMed Scopus (123) Google Scholar, 5Smith W.W. Pattridge K.A. Ludwig M.L. Petsko G.A. Tsernoglou D. Tanaka M. Yasunobu K.T. J. Mol. Biol. 1983; 165: 737-753Crossref PubMed Scopus (83) Google Scholar, 6Rao S.T. Shaffie F. Yu C. Satyshur K.A. Stockman B.J. Marley J.L. Sundaralingam M. Protein Sci. 1992; 1: 1413-1427Crossref PubMed Scopus (100) Google Scholar), the structural difference was seen to be due to the presence in long flavodoxins of an extra loop that splits the fifth strand of the central β-sheet (Fig. 1). Because many of the functional and thermodynamic properties of short and long flavodoxins are similar (redox potentials, affinity for the FMN redox cofactor, and so forth), it is not clear yet what role the extra loop of the long flavodoxins may play. In our laboratory, we have used the holoform (6Rao S.T. Shaffie F. Yu C. Satyshur K.A. Stockman B.J. Marley J.L. Sundaralingam M. Protein Sci. 1992; 1: 1413-1427Crossref PubMed Scopus (100) Google Scholar) and apoform (7Genzor C.G. Perales-Alcon A. Sancho J. Romero A. Nat. Struct. Biol. 1996; 3: 329-332Crossref PubMed Scopus (82) Google Scholar) of the flavodoxin from Anabaena as models to investigate protein folding (8Fernández-Recio J. Genzor C.G. Sancho J. Biochemistry. 2001; 40: 15234-15245Crossref PubMed Scopus (46) Google Scholar), protein stability (9Genzor C.G. Beldarrain A. Gomez-Moreno C. Lopez-Lacomba J.L. Cortijo M. Sancho J. Protein Sci. 1996; 5: 1376-1388Crossref PubMed Scopus (72) Google Scholar, 10Fernández-Recio J. Romero A. Sancho J. J. Mol. Biol. 1999; 290: 319-330Crossref PubMed Scopus (65) Google Scholar, 11Langdon G.M. Jimenez M.A. Genzor C.G. Maldonado S. Sancho J. Rico M. Proteins. 2001; 43: 476-488Crossref PubMed Scopus (27) Google Scholar, 12Irun M.P. Garcia-Mira M.M. Sánchez-Ruiz J.M. Sancho J. J. Mol. Biol. 2001; 306: 877-888Crossref PubMed Scopus (54) Google Scholar, 13Irun M.P. Maldonado S. Sancho J. Protein Eng. 2001; 14: 173-181Crossref PubMed Scopus (32) Google Scholar, 14Maldonado S. Lostao A. Irún M.P. Fernández-Recio J. Genzor C.G. González E.B. Rubio J.A. Luquita A. Daoudi F. Sancho J. Biochimie (Paris). 1998; 80: 813-820Crossref PubMed Scopus (17) Google Scholar, 15Maldonado S. Jimenez M.A. Langdon G.M. Sancho J. Biochemistry. 1998; 37: 10589-10596Crossref PubMed Scopus (32) Google Scholar, 16Maldonado S. Irun M.P. Campos L.A. Rubio J.A. Luquita A. Lostao A. Wang R. Garcia-Moreno E.B. Sancho J. Protein Sci. 2002; 11: 1260-1273Crossref PubMed Scopus (24) Google Scholar), and protein/ligand interaction (17Lostao A. Gómez-Moreno C. Mayhew S.G. Sancho J. Biochemistry. 1997; 36: 14334-14344Crossref PubMed Scopus (81) Google Scholar, 18Lostao A. El Harrous M. Daoudi F. Romero A. Parody-Morreale A. Sancho J. J. Biol. Chem. 2000; 275: 9518-9526Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 19Lostao A. Daoudi F. Irún M.P. Ramón A. Fernández-Cabrera C. Romero A. Sancho J. J. Biol. Chem. 2003; 278: 24053-24061Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 20Casaus J.L. Navarro J.A. Hervas M. Lostao A. De La Rosa M.A. Gomez-Moreno C. Sancho J. Medina M. J. Biol. Chem. 2002; 277: 22338-22344Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) and a wealth of thermodynamic and kinetic information is now available on this protein. In this work, we investigate the influence of the long loop of Anabaena flavodoxin on the structure and cofactor binding of the apoprotein by deriving and studying two shortened variants where the long loop has been removed by site-directed mutagenesis. Although one of the shortened variants displays a somewhat altered structure compatible with some local unfolding, the second one is clearly well folded, indicating that the loop is not required for the correct folding of the long flavodoxins. Surprisingly, the affinity of the shortened protein for its cofactor is severely reduced despite the fact that the loop makes no contacts with the FMN in the structure of the holoflavodoxin. From a comparison of the structures of short and long flavodoxins, we propose that this could be related to a role played by the long loop in stabilizing the native conformation of the adjacent tyrosine-bearing loop involved in FMN binding. Additionally, a structural analysis of the conformation of the fifth β-strand in short and long flavodoxins points to the possibility that short flavodoxins derive from the long ones. Site-directed Mutagenesis, Protein Expression, Purification, and Quantitation—Oligonucleotide-directed mutagenesis of the flavodoxin gene cloned in the plasmid pTrc99a (21Amann E. Ochs B. Abel K.J. Gene (Amst.). 1998; 69: 310-315Google Scholar) was performed by a modification of the method of Deng and Nickoloff (22Deng W.P. Nickoloff J.A. Anal. Biochem. 1992; 200: 81-88Crossref PubMed Scopus (1078) Google Scholar). The mutagenic oligonucleotide used to delete residues 119–139 to produce the shorter mutant flavodoxin, Δ(119–139), with Gly118 adjacent to Gly140 was 5′-GATTATCTTCATCAAGAGCTAGTCCGCCGACAGTTTTACCACCAC-3′. To delete residues 120–139 and produce Δ(120–139) with Tyr119 adjacent to Gly140, the oligonucleotide used was 5′-GATTATCTTCATCAAGAGCTAGTCCATAGCCGACAGTTTTACCAC-3′. Mutant plasmids were identified by direct sequencing. The expression and purification of flavodoxin mutants were done essentially as described for the wild type protein (23Fillat M.F. Borrias W.E. Weisbeek P. Biochem. J. 1991; 280: 187-191Crossref PubMed Scopus (78) Google Scholar). The purity of each flavodoxin preparation was confirmed by SDS-polyacrylamide gel electrophoresis. The mutants were purified as apoproteins, as they lost the FMN prosthetic group along the purification. The concentration of the mutants was determined from the absorbance at 280 nm (24Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5010) Google Scholar) using an extinction coefficient of 25,590 m-1 cm-1 for Δ(119–139) and of 27,000 m-1 cm-1 for Δ(120–139). Absorbance, Fluorescence, Circular Dichroism, and1H NMR Spectra—Absorbance spectra were recorded at 298.2 ± 0.1 K in a Kontron Uvikon 860 spectrophotometer. Corrected steady-state fluorescence emission at 298.2 ± 0.1 K in the 300–450-nm range was obtained on a SLM 8000D spectrofluorometer with excitation at 295 nm. Far-UV and near-UV circular dichroism spectra at 298.2 ± 0.1 K were recorded in a Jasco 710 spectropolarimeter using a 0.1- and 1-cm cuvette, respectively. 1H NMR spectra were acquired on a Bruker AMX-600 pulse spectrometer operating at a proton frequency of 600.13 MHz. One-dimensional spectra were recorded at 298.2 ± 0.1 K using 32-K data points zero-filled to 64 K before performing the was by The of the NMR was with for NMR were at protein concentration in was the of the of Δ(119–139) and of Δ(119–139) and Δ(120–139) were determined by in protein with a in The was FMN to Δ(120–139) interaction of the FMN cofactor with Δ(120–139) was binding of FMN to its fluorescence and by The FMN used was according to The fluorescence were in a Kontron at emission from to at 298.2 ± 0.1 K in the The cofactor of FMN in was with 0.1 of Δ(120–139) in the and the was to for before an emission was In the an protein was with a in The was and the absorbance at nm was of Δ(120–139) and FMN in the for was in the and fluorescence and at 298.2 ± 0.1 K were performed in using the The and analysis was similar to the one described M.P. Biochemistry. 2002; PubMed Scopus Google Scholar). fluorescence at emission were a were described by a of the analysis be described fluorescence a direct of the hydrodynamic of proteins in the are related to and by the of Δ(119–139) and Δ(120–139) spectroscopic and hydrodynamic properties of Δ(119–139) and Δ(120–139) have been with of the protein. Because both the fluorescence and near-UV spectra of wild type by from M.P. Maldonado S. Sancho J. Protein Eng. 2001; 14: 173-181Crossref PubMed Scopus (32) Google Scholar) and this is no longer in the shortened flavodoxins, we the spectroscopic properties of the shortened with of the mutant wild used wild which is a more than wild We have the fluorescence emission spectra of the shortened variants with that of (Fig. The mutants a more the Δ(120–139) and in the emission spectra at nm to The determined at emission and the fluorescence were used to the emission spectra with The spectra B. P. P. J. Chem. Scopus Google Scholar) in of the the not The of the emission spectra with the for and for both was nm. This has been to P. and J. Sancho, in is the one that to the for the protein The of residues to in the shortened and in the has been by absorbance difference spectra in the The of the difference spectra (Fig. is the in the indicating a in of The two similar difference the of the shorter Δ(119–139) is a which is of a of its residues in the The and near-UV spectra of the short flavodoxins are with of in and respectively. The spectra indicate that the two shortened structure with not from that of The of to is due to its reduced of loop In its is the for Δ(119–139), its is that of and its and of nm that it is somewhat This is by the near-UV data (Fig. both and Δ(120–139) spectra in this indicating that they some well interactions Δ(119–139) a from However, in a protein with this is not of a of well we have acquired 1H NMR spectra of the two shortened (Fig. The of the and the presence of at and in the clearly indicate that the two shortened variants are well and to a of local interactions in Δ(119–139) than to a more of Δ(119–139) and Δ(120–139) to the spectroscopic have been mutant flavodoxins are from the gel as not are from to This that the flavodoxins are The than that of indicate that the shortened proteins are as the protein as as gel From a of the we the difference in the shortened and the at the difference To a of the of the variants in we have determined their which are on their hydrodynamic Because the variants their fluorescence emission be by local of each and by the protein To the of to the we have performed a fluorescence and of the wild type protein and of a of The and of the variants have been from their The for data not well with that determined for wild type and with the from the a of from the x-ray structure of the wild type The for Δ(120–139) is the the In Δ(119–139) displays a of than that of which that the structure of Δ(119–139) is than that of Δ(120–139). FMN to Δ(120–139) Δ(119–139) and Δ(120–139) are purified as apoproteins, the FMN prosthetic group lost in the purification. We have to the holoform of the Δ(120–139) by FMN to the binding of the cofactor was at a a in the emission of FMN that from to was not This that FMN to Δ(120–139) that the affinity is than in the wild type protein. To a for the affinity of the we performed where a of FMN and Δ(120–139) for was a absorbance at nm were (Fig. one to the of FMN and a second one to the of Δ(120–139). of binding from is the that is may the the is for the be that no has in the and that the of the species from the absorbance of their in the The fact that the of the in the of the are similar to of the that the is not in this From the absorbance of the we that is than m-1 to an affinity of the than are obtained the of the than their are used in the not The affinity of the wild type is A. El Harrous M. Daoudi F. Romero A. Parody-Morreale A. Sancho J. J. Biol. Chem. 2000; 275: 9518-9526Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) at the the of the of the long flavodoxins, the of the long loop splitting the fifth strand is as it is the of the of the protein not This that the loop may a role from the structural or the functional of To the structural of the we have constructed two shortened where the Gly118 or the Tyr119 wild type has been to the wild type Gly140 thus the long loop from the Anabaena The expression of the two variants were and shortened in the S. Jimenez M.A. Langdon G.M. Sancho J. Biochemistry. 1998; 37: 10589-10596Crossref PubMed Scopus (32) Google Scholar), they be purified in the used to wild type they are obtained in the The hydrodynamic performed by that both shortened are and that their are with of than markedly ones. is by the fluorescence emission spectra and the near-UV absorbance difference spectra (Fig. and that that the the are not to In addition, both shortened a structure (Fig. However, are that indicate that Δ(119–139) is somewhat its absorbance difference is its is and its near-UV spectra are with this spectroscopic our analysis of the that, Δ(120–139) and are Δ(119–139) is somewhat we that, in the NMR spectra (Fig. both shortened and similar to of well This that Δ(119–139) is in a not from the one described for the wild type protein (7Genzor C.G. Perales-Alcon A. Sancho J. Romero A. Nat. Struct. Biol. 1996; 3: 329-332Crossref PubMed Scopus (82) Google Scholar, C.G. Beldarrain A. Gomez-Moreno C. Lopez-Lacomba J.L. Cortijo M. Sancho J. Protein Sci. 1996; 5: 1376-1388Crossref PubMed Scopus (72) Google Scholar), and the and of near-UV in Δ(119–139) may be related to the local of a of the an The the two variants were may to their structural were to for the protein to the removal of the loop. In the of Gly140 is at similar from the of Gly118 and of Tyr119 To a β-strand of the two and and it with strand only one is In this respect, the shorter mutant Δ(119–139) of the residues to a fifth strand of the The fact that it to a to native conformation is the of an of the fifth β-strand by residues and This makes the shortened protein a conformation as from the reduced one is and the long loop are the In the spectroscopic properties of the longer Δ(120–139) that the is to a The by the extra the mutant protein to a native In the of the available x-ray structures of short flavodoxins Desulfovibrio Protein and from Clostridium Protein that their fifth β-strand a at the where it split by the long loop in the long flavodoxins. the correct folding of the shortened Δ(120–139) clearly the of the extra loop of long flavodoxins for a well this long loop to a peripheral role from the structural of The the and FMN have the interaction of FMN with shortened flavodoxin using the Δ(120–139) which is the affinity of Δ(120–139) for the prosthetic group is despite the fact that of the residues of the loop direct contact with FMN in holoflavodoxin. be that long loop residues are in contact (Fig. with the short loop that the and in Anabaena that on one of the FMN in the of flavodoxins of the known structure (17Lostao A. Gómez-Moreno C. Mayhew S.G. Sancho J. Biochemistry. 1997; 36: 14334-14344Crossref PubMed Scopus (81) Google Scholar) and the functional A. El Harrous M. Daoudi F. Romero A. Parody-Morreale A. Sancho J. J. Biol. Chem. 2000; 275: 9518-9526Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). is clear that the loop could produce the in the affinity for The fact that the fluorescence of at the loop involved in FMN binding and is altered in the shortened may a of conformational to this more FMN binding loop. we that, in short flavodoxins, the loop is shorter than in long flavodoxins one or two see This in short flavodoxins the conformational of the loop to a that no interactions with the long loop be to the of the redox and FMN binding in its functional the long loop not be as an feature for the binding of the flavodoxins that it to the loop to long flavodoxins (Fig. and FMN the long loop seems to be used by long flavodoxins as a conformational to the FMN binding competent conformation of their longer thus the affinity of the functional complex. is in in our to to the FMN affinity of Δ(120–139) by its loop by of short flavodoxins. on a of Flavodoxins from the it is not clear which of the flavodoxin gene long or the short is To investigate this we have performed a analysis of the available short and long flavodoxin not The analysis that transfer of the flavodoxin gene has been which the However, from a we have a structural feature of the short-chain flavodoxins that be with their from the long ones. has been the fifth β-strand of short flavodoxins a at the where the loop of the long flavodoxins from the We in a of the of the long Anabaena flavodoxin and of the short flavodoxin from Desulfovibrio The are similar and be in the short flavodoxin, the and is clearly the fifth strand a The short flavodoxin from Clostridium the at the not We that the is where the long flavodoxins the long loop that split their fifth In it is that an short flavodoxins have a central β-sheet that was in of them in the presence of a and that the long loop was at the we more that the a of the lost loop of the more long flavodoxins. for a of the presence of this long loop in so many flavodoxins and the fact that its is as that of the of the protein not with the influence of the loop in the structure and stability of the protein J. Maldonado S. S. Lostao A. R. Cortijo M. Fernández-Recio J. Sancho J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google for that the loop could an functional role than a structural for we have performed a for short and of the loop in no of the loop are not the the of the loop in the not from the redox of the where the FMN group is bound (Fig. we that one role of the loop could be that of binding residues for flavodoxin of these have been identified in the where the flavodoxins are In flavodoxin is required for the of C. J. 1991; PubMed Google Scholar), for K.J. Biochem. 1996; PubMed Scopus (82) Google Scholar), and for the of both V. M. E. P. Biochem. PubMed Scopus Google Scholar) and G. G. Mol. 1998; PubMed Scopus Google Scholar). In as flavodoxin is the electron for the protein M.F. G. Gomez-Moreno C. Scopus Google Scholar). In addition, flavodoxins and have been to be of the of Biochem. PubMed Scopus Google Scholar) and of E. G. M. J. J. Biochem. PubMed Scopus Google Scholar), of the of Clostridium M.P. Biochem. J. PubMed Scopus Google Scholar), and of the of J. R. Biol. Sci. PubMed Scopus Google Scholar). that the flavodoxin long loop could be involved in the interaction of E. flavodoxin with has been by NMR Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). to the flavodoxin binding to residues of the flavodoxin long loop could be in contact with the in the complex. In the the was investigated. a of loop residues or were to their binding to In in the the long loop is the flavodoxin where the Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). it seems that the flavodoxin long loop could an role in the recognition of non-photosynthetic In addition, our analysis not that photosynthetic is to long flavodoxin a possibility that the flavodoxin long loop is involved in the recognition of photosynthetic Although the loop splitting the fifth β-strand in long-chain flavodoxins not FMN binding residues and it is not for the folding of the it seems to the adjacent FMN binding loop to the that Anabaena no longer FMN with affinity the long loop is This not be to that the long loop role is to in FMN binding, the short flavodoxins FMN the conservation of the long loop flavodoxins and its influence on the structure and stability of the protein J. Maldonado S. S. Lostao A. R. Cortijo M. Fernández-Recio J. Sancho J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), we that it to with flavodoxin partners.

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