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PubMed1997

Three-dimensional structure of NADPH-cytochrome P450 reductase: prototype for FMN- and FAD-containing enzymes.

Microsomal NADPH-cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat liver CPR, expressed in Escherichia coli and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 A resolution. The molecule is composed of four structural domains: (from the N- to C- termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains. The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin-NADP+ reductase (FNR). The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer. The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 A. The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome c and the physiological redox partners, including cytochromes P450 and b5 and heme oxygenase.

Amino Acid Sequence↗
PubMed2003

How FMN binds to anabaena apoflavodoxin: a hydrophobic encounter at an open binding site.

Molecular recognition begins when two molecules approach and establish interactions of certain strength. The mechanisms of molecular recognition reactions between biological molecules are not well known, and few systems have been analyzed in detail. We investigate here the reaction between an apoprotein and its physiological cofactor (apoflavodoxin and flavin mononucleotide) that binds reversibly to form a non-covalent complex (flavodoxin) involved in electron transfer reactions. We have analyzed the fast binding reactions between the FMN cofactor (and shorter analogs) and wild type (and nine mutant apoflavodoxins where residues interacting with FMN in the final complex have been replaced). The x-ray structures of two such mutants are reported that show the mutations are well tolerated by the protein. From the calculated microscopic binding rate constants we have performed a Phi analysis of the transition state of complex formation that indicates that the binding starts by interaction of the isoalloxazine-fused rings in FMN with residues of its hydrophobic binding site. In contrast, the phosphate in FMN, known to contribute most to the affinity of the final holoflavodoxin complex, is not bound in the transition state complex. Both the effects of ionic strength and of phosphate concentration on the wild type complex rate constant agree with this scenario. As suggested previously by nmr data, it seems that the isoalloxazine-binding site may be substantially open in solution. Interestingly, although FMN is a charged molecule, electrostatic interactions seem not to play a role in directing the binding, unlike what has been reported for other biological complexes. The binding can thus be best described as a hydrophobic encounter at an open binding site.

Anabaena↗
PubMed2005

An FMN hydrolase is fused to a riboflavin kinase homolog in plants.

Riboflavin kinases catalyze synthesis of FMN from riboflavin and ATP. These enzymes have to date been cloned from bacteria, yeast, and mammals, but not from plants. Bioinformatic approaches suggested that diverse plant species, including many angiosperms, two gymnosperms, a moss (Physcomitrella patens), and a unicellular green alga (Chlamydomonas reinhardtii), encode proteins that are homologous to riboflavin kinases of yeast and mammals, but contain an N-terminal domain that belongs to the haloacid dehalogenase superfamily of enzymes. The Arabidopsis homolog of these proteins was cloned by RT-PCR, and was shown to have riboflavin kinase and FMN hydrolase activities by characterizing the recombinant enzyme produced in Escherichia coli. Both activities of the purified recombinant Arabidopsis enzyme (AtFMN/FHy) increased when the enzyme assays contained 0.02% Tween 20. The FMN hydrolase activity of AtFMN/FHy greatly decreased when EDTA replaced Mg(2+) in the assays, as expected for a member of the Mg(2+)-dependent haloacid dehalogenase family. The functional overexpression of the individual domains in E. coli establishes that the riboflavin kinase and FMN hydrolase activities reside, respectively, in the C-terminal (AtFMN) and N-terminal (AtFHy) domains of AtFMN/FHy. Biochemical characterization of AtFMN/FHy, AtFMN, and AtFHy shows that the riboflavin kinase and FMN hydrolase domains of AtFMN/FHy can be physically separated, with little change in their kinetic properties.

Amino Acid Sequence↗
PubMed2006

Surface charge interactions of the FMN module govern catalysis by nitric-oxide synthase.

The FMN module of nitric-oxide synthase (NOS) plays a pivotal role by transferring NADPH-derived electrons to the enzyme heme for use in oxygen activation. The process may involve a swinging mechanism in which the same face of the FMN module accepts and provides electrons during catalysis. Crystal structure shows that this face of the FMN module is electronegative, whereas the complementary interacting surface is electropositive, implying that charge interactions enable function. We used site-directed mutagenesis to investigate the roles of six electronegative surface residues of the FMN module in electron transfer and catalysis in neuronal NOS. Results are interpreted in light of crystal structures of NOS and related flavoproteins. Neutralizing or reversing the negative charge of each residue altered the NO synthesis, NADPH oxidase, and cytochrome c reductase activities of neuronal NOS and also altered heme reduction. The largest effects occurred at the NOS-specific charged residue Glu(762). Together, the results suggest that electrostatic interactions of the FMN module help to regulate electron transfer and to minimize flavin autoxidation and the generation of reactive oxygen species during NOS catalysis.

Animals↗
PubMed1975

Properties of immobilized flavodoxin from Peptostreptococcus elsdenii. An affinity ligand for the purification of riboflavin 5'-phosphate (FMN) and its analogues.

The small flavoprotein, flavodoxin, isolated from Peptostreptococcus elsdenii, has been covalently coupled to CNBr-activated Sepharose 4B. The immobilized protein replaces ferredoxin as an electron carrier in hydrogen production from dithionite or pyruvate in the presence of ferredoxin-free extracts of P. elsdenii; compared with soluble flavodoxin, its activities in these systems are 13% and 3.5% respectively. Acid treatment reversibly dissociates FMN from the immobilized protein. The dissociation constant of the complex with FMN, determined by fluorimetric titration, is 1.5 (+/- 0.4) nM, and is therefore very little different from that of soluble flavodoxin. Like soluble apoflavodoxin, the immobilized apoprotein is highly specific for flavins with an N-10 side-chain of 5 carbon atoms and a C-5' phosphate group. Approximately half of the flavin impurity in commercial preparations of FMN (12-15% of the total flavin), and similar impurity in synthetic analogues of FMN, is not separated by conventional purification procedures, but it is readily and conveniently removed by affinity chromatography with apoflavodoxin as the immobilized ligand. The immobilized protein is stable for long periods; its capacity for FMN decreases by only 20% after 15 cycles of flavin dissociation and reassociation during several months.

Bacterial Proteins↗
PubMed1983

2-thioriboflavin 5'-phosphate (2-thio-FMN) lactate oxidase.

The natural flavin of lactate oxidase, FMN, was removed and replaced by the synthetic flavin 2-thioriboflavin 5'-phosphate (2-thio FMN). Despite the difference in properties of the flavins, including an oxidation-reduction potential some 80 mV more positive than that of normal flavin the 2-thio-FMN enzyme behaves in practically all respects like the native enzyme. It catalyzes the oxidative decarboxylation of L-lactate with almost the same efficiency as the native enzyme and with similar kinetic constants for individual steps in the catalytic pathway. It forms covalent derivatives at the flavin N(5) and C(4a) positions in facile photochemical reactions analogous to those of the native enzyme. It also forms a flavin anion radical on photoreduction with 5-deazaflavin as catalyst and, as with the native enzyme, this radical is stabilized remarkably on formation of a complex with pyruvate. The spectral properties of the neutral flavin radical form of 2-thioflavin are also reported, as determined by photochemical reduction of 2-thio-FMN flavodoxin. Like native lactate oxidase, the 2-thio-FMN enzyme also forms a flavin N(5)-sulfite adduct in an equilibrium reaction with sulfite. These results demonstrate clearly with this enzyme that the native flavin may be removed and replaced by an artificial flavin, without altering the structural integrity of the protein.

Anaerobiosis↗
PubMed1991

Studies on the flavin-binding region of old yellow enzyme with an active site probe, 8-fluoro-8-demethyl FMN.

The brewer's yeast old yellow enzyme (OYE) was reconstituted with 8-fluoro-8-demethyl FMN (8F-FMN). The reconstituted enzyme exhibited absorption maxima at 355 and 450 nm in the visible region. This reconstituted enzyme underwent no further spectral changes, showing no evidence of modification in the flavin moiety. However, when the reconstituted enzyme was subjected to specific limited proteolysis with bovine alpha-chymotrypsin, gradual spectral changes were observed with disappearance of the 355- and 450-nm bands accompanied by the appearance of a new band at 496 nm. Identical spectral changes were observed when the proteolytically cleaved OYE (nicked OYE) was reconstituted with 8F-FMN. The process associated with these spectral changes was found to be unimolecular by kinetic analysis. Reverse-phase HPLC analysis revealed that these spectral changes resulted from covalent bond formation between 8F-FMN and the protein moiety after the proteolytic cleavage of the protein into 14K and 34K fragments. The reverse-phase HPLC monitored at 490 nm showed that the chromophore with 496 nm absorption maximum was covalently attached to the 14K fragment. The amino acid sequence analysis of the flavinylated 14K fragment together with that of the 14K fragment of native OYE indicated that the N-terminal leucine of the 14K fragment is the site of flavinylation. These findings imply that the amino group of the N-terminal leucine of the 14K fragment became available as the result of proteolysis and that this amino group nucleophilically attacked the 8-position of 8F-FMN, forming a covalent bond between the flavin moiety and the 14K fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1982

Oxidation-reduction states of FMN and FAD in NADPH-cytochrome P-450 reductase during reduction by NADPH.

NADPH-cytochrome P-450 reductase, a component of the multisubstrate monooxygenase system of liver microsomes, is an unusual flavoprotein in that it contains both FMN and FAD. In recent studies in this laboratory, a procedure was devised for selective removal of FMN from the purified enzyme, thus leading to the identification of FMN and FAD as the prosthetic groups of high and low reduction potential, respectively, and to the assignment of known reduction potentials to the individual flavin half-reactions. In the present study, the reaction of NADPH with the reductase was examined under anaerobic conditions by stopped flow spectrophotometry. The results were shown to correspond to those predicted on the basis of a model for the rapid exchange of reducing equivalents between the two flavins, the distribution being governed at any time by the reduction potentials for the individual flavin half-reactions. The reaction is divided into three steps, as follows (a) In a rapid first phase with a first order rate constant of 28 s-1, a mixture of about 70% (FMNH2, FAD) and 30% disemiquinone (FMNH ., FADH .) is generated; (FMN, FADH2), the presumed transient intermediate in the reduction of the oxidized flavoprotein by NADPH, does not accumulate under these conditions. (b) In a second phase characterized by a first order rate constant of 5.4 s-1, a mixture of 65% (FMNH2, FADH2), 24% (FMNH2, FAD), and 11% (FMNH ., FADH .) is produced. (c) Regardless of the NADPH concentration employed, a third phase occurs with very slow changes leading to an equilibrium mixture of the nine oxidation-reduction states of the reductase. The absorption spectra for all possible oxidation-reduction states of the FMN moiety of the reductase as well as of the native reductase are presented.

Cytochromes↗
PubMed1994

The turnover of bacterial luciferase is limited by a slow decomposition of the ternary enzyme-product complex of luciferase, FMN, and fatty acid.

Bacterial luciferase catalyzes the conversion of reduced flavin mononucleotide, O2, and fatty aldehyde to FMN, H2O, and fatty acid with light being emitted at a very low rate characterized by the decay of luminescence in single turnover flash assays. The present studies have now revealed that the decomposition of the ternary complex of luciferase with FMN and myristic acid occurs at a rate 10-15 times slower than the decay of luminescence and that functional luciferase is only regenerated after the release of flavin. In contrast, the rate of FMN dissociation and recovery of activity observed with a binary luciferase-FMN complex was more rapid indicating that release of fatty acid played the critical role in determining the rate of FMN dissociation from the ternary complex. Decomposition of the ternary complex was shown to occur by an ordered process involving the slow release of fatty acid followed by the more rapid release of flavin. The present results suggest that the rate-limiting step for the turnover of luciferase occurs subsequent to emission of light and would be the controlling step under conditions (e.g. in cells) with continuous light emission.

Enzyme Stability↗
PubMedDate not supplied

Characteristics of endogenous flavin fluorescence of Photobacterium leiognathi luciferase and Vibrio fischeri NAD(P)H:FMN-oxidoreductase.

The bioluminescent bacterial enzyme system NAD(P)H:FMN-oxidoreductase-luciferase has been used as a test system for ecological monitoring. One of the modes to quench bioluminescence is the interaction of xenobiotics with the enzymes, which inhibit their activity. The use of endogenous flavin fluorescence for investigation of the interactions of non-fluorescent compounds with the bacterial luciferase from Photobacterium leiognathi and NAD(P)H:FMN-oxidoreductase from Vibrio fischeri has been proposed. Fluorescence spectroscopy methods have been used to study characteristics of endogenous flavin fluorescence (fluorophore lifetime, the rotational correlation time). The fluorescence anisotropy behaviour of FMN has been analysed and compared to that of the enzyme-bound flavin. The fluorescence characteristics of endogenous flavin of luciferase and NAD(P)H:FMN-oxidoreductase have been shown to be applicable in studying enzymes' interactions with non-fluorescent compounds.

Aliivibrio fischeri↗
PubMed2000

Vibrio harveyi NADPH-FMN oxidoreductase arg203 as a critical residue for NADPH recognition and binding.

Luminous bacteria contain three types of NAD(P)H-FMN oxidoreductases (flavin reductases) with different pyridine nucleotide specificities. Among them, the NADPH-specific flavin reductase from Vibrio harveyi exhibits a uniquely high preference for NADPH. In comparing the substrate specificity, crystal structure, and primary sequence of this flavin reductase with other structurally related proteins, we hypothesize that the conserved Arg203 residue of this reductase is critical to the specific recognition of NADPH. The mutation of this residue to an alanine resulted in only small changes in the binding and reduction potential of the FMN cofactor, the K(m) for the FMN substrate, and the k(cat). In contrast, the K(m) for NADPH was increased 36-fold by such a mutation. The characteristic perturbation of the FMN cofactor absorption spectrum upon NADP(+) binding by the wild-type reductase was abolished by the same mutation. While the k(cat)/K(m,NADPH) was reduced from 1990 x 10(5) to 46 x 10(5) M(-1) min(-1) by the mutation, the mutated variant showed a k(cat)/K(m,NADH) of 4 x 10(5) M(-1) min(-1), closely resembling that of the wild-type reductase. The deuterium isotope effects (D)V and (D)(V/K) for (4R)-[4-(2)H]-NADPH were 1.7 and 1.4, respectively, for the wild-type reductase but were increased to 3.8 and 4.0, respectively, for the mutated variant. Such a finding indicates that the rates of NADPH and NADP(+) dissociation in relation to the isotope-sensitive redox steps were both increased as a result of the mutation. These results all provide support to the critical role of the Arg203 in the specific recognition and binding of NADPH.

Amino Acid Sequence↗
PubMed2004

Crystal structure and functional characterization of yeast YLR011wp, an enzyme with NAD(P)H-FMN and ferric iron reductase activities.

Flavodoxins are involved in a variety of electron transfer reactions that are essential for life. Although FMN-binding proteins are well characterized in prokaryotic organisms, information is scarce for eukaryotic flavodoxins. We describe the 2.0-A resolution crystal structure of the Saccharomyces cerevisiae YLR011w gene product, a predicted flavoprotein. YLR011wp indeed adopts a flavodoxin fold, binds the FMN cofactor, and self-associates as a homodimer. Despite the absence of the flavodoxin key fingerprint motif involved in FMN binding, YLR011wp binds this cofactor in a manner very analogous to classical flavodoxins. YLR011wp closest structural homologue is the homodimeric Bacillus subtilis Yhda protein (25% sequence identity) whose homodimer perfectly superimposes onto the YLR011wp one. Yhda, whose function is not documented, has 53% sequence identity with the Bacillus sp. OY1-2 azoreductase. We show that YLR011wp has an NAD(P)H-dependent FMN reductase and a strong ferricyanide reductase activity. We further demonstrate a weak but specific reductive activity on azo dyes and nitrocompounds.

Amino Acid Sequence↗
PubMed1995

Cloning of FAD synthetase gene from Corynebacterium ammoniagenes and its application to FAD and FMN production.

The cloning of a bifunctional FAD synthetase gene, which shows flavokinase and FMN adenylyltransferase activities, from Corynebacterium ammoniagenes was tried by hybridization with synthetic DNAs corresponding to the N-terminal amino acid sequence. The cloned PstI-digested 4.4 x 10(3)-base (4.4-kb) fragment could not express the FAD synthetase activity in E. coli, but could increase the two activities by the same factor of about 20 in C. ammoniagenes. The FAD-synthetase-gene-amplified C. ammoniagenes cells were applied to the production of FAD from FMN or riboflavin. The productivity of FAD from FMN was increased four to five times compared with the parent strain, and reached a 90% molar yield. The productivity of FAD from riboflavin was increased about eight times, with a 50% molar yield. The addition of Zn2+ to the reaction mixtures for the conversion from riboflavin to FAD brought about the specific inhibition of adenylyl-transferase activity and resulted in the accumulation of FMN.

Amino Acid Sequence↗
PubMed2005

The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch.

Riboswitches are genetic control elements that usually reside in untranslated regions of messenger RNAs. These folded RNAs directly bind metabolites and undergo allosteric changes that modulate gene expression. A flavin mononucleotide (FMN)-dependent riboswitch from the ribDEAHT operon of Bacillus subtilis uses a transcription termination mechanism wherein formation of an RNA-FMN complex causes formation of an intrinsic terminator stem. We assessed the importance of RNA transcription speed and the kinetics of FMN binding to the nascent mRNA for riboswitch function. The riboswitch does not attain thermodynamic equilibrium with FMN before RNA polymerase needs to make a choice between continued transcription and transcription termination. Therefore, this riboswitch is kinetically driven, and functions more like a "molecular fuse." This reliance on the kinetics of ligand association and RNA polymerization speed might be common for riboswitches that utilize transcription termination mechanisms.

Bacillus subtilis↗
PubMed1997

The self-association of flavin mononucleotide (FMN(2-)) as determined by (1)H NMR shift measurements.

The concentration dependence of the (1)H NMR chemical upfield shifts of the protons H6, H9, H7alpha, and H8alpha of the 7,8-dimethylisoalloxazine residue of flavin mononucleotide (FMN(2-)) has been measured and the self-stacking tendency of FMN(2-) was quantified with the isodesmic model of indefinite non-cooperative self-association. The stacking tendency of FMN(2-) is considerable and described in the concentration range of 0.0025-0.1 M with the indicated model by K = 27 +/- 15 M(-1) (25 degrees C; I = 0.1-0.3 M). This result is compared with related ones from the literature. The caveats regarding the self-stacking properties of FMN(2-) and their dependence on the concentration are discussed.

Journal Article↗
PubMed2000

The X-ray structure of the FMN-binding protein AtHal3 provides the structural basis for the activity of a regulatory subunit involved in signal transduction.

BACKGROUND: The Arabidopsis thaliana HAL3 gene product encodes for an FMN-binding protein (AtHal3) that is related to plant growth and salt and osmotic tolerance. AtHal3 shows sequence homology to ScHal3, a regulatory subunit of the Saccharomyces cerevisae serine/threonine phosphatase PPz1. It has been proposed that AtHal3 and ScHal3 have similar roles in cellular physiology, as Arabidopsis transgenic plants that overexpress AtHal3 and yeast cells that overexpress ScHal3 display similar phenotypes of improved salt tolerance. The enzymatic activity of AtHal3 has not been investigated. However, the AtHal3 sequence is homologous to that of EpiD, a flavoprotein from Staphylococcus epidermidis that recognizes a peptidic substrate and subsequently catalyzes the alpha, beta-dehydrogenation of its C-terminal cysteine residue. RESULTS: The X-ray structure of AtHal3 at 2 A resolution reveals that the biological unit is a trimer. Each protomer adopts an alpha/beta Rossmann fold consisting of a six-stranded parallel beta sheet flanked by two layers of alpha helices. The FMN-binding site of AtHal3 contains all the structural requirements of the flavoenzymes that catalyze dehydrogenation reactions. Comparison of the amino acid sequences of AtHal3, ScHal3 and EpiD reveals that a significant number of residues involved in trimer formation, the active site, and FMN binding are conserved. This observation suggests that ScHal3 and EpiD might also be trimers, having a similar structure and function to AtHal3. CONCLUSIONS: Structural comparisons of AtHal3 with other FMN-binding proteins show that AtHal3 defines a new subgroup of this protein family that is involved in signal transduction. Analysis of the structure of AtHal3 indicates that this protein is designed to interact with another cellular component and to subsequently catalyze the alpha,beta-dehydrogenation of a peptidyl cysteine. Structural data from AtHal3, together with physiological and biochemical information from ScHal3 and EpiD, allow us to propose a model for the recognition and regulation of AtHal3/ScHal3 cellular partners.

Amino Acid Sequence↗
PubMed2001

Determination of the midpoint potential of the FAD and FMN flavin cofactors and of the 3Fe-4S cluster of glutamate synthase.

Glutamate synthase is a complex iron-sulfur flavoprotein that catalyzes the reductive transfer of the L-glutamine amide group to C(2) of 2-oxoglutarate, forming two molecules of L-glutamate. The bacterial enzyme is an alphabeta protomer, which contains one FAD (on the beta subunit, approximately 50 kDa), one FMN (on the alpha subunit, approximately 150 kDa), and three different Fe-S clusters (one 3Fe-4S center on the alpha subunit and two 4Fe-4S clusters at an unknown location). To address the problem of the intramolecular electron pathway, we have measured the midpoint potential values of the flavin cofactors and of the 3Fe-4S cluster of glutamate synthase in the isolated alpha and beta subunits and in the alphabeta holoenzyme. No detectable amounts of flavin semiquinones were observed during reductive titrations of the enzyme, indicating that the midpoint potential value of each flavin(ox)/flavin(sq) couple is, in all cases, significantly more negative than that of the corresponding flavin(sq)/flavin(hq) couple. Association of the two subunits to form the alphabeta protomer does not alter significantly the midpoint potential value of the FMN cofactor and of the 3Fe-4S cluster (approximately -240 and -270 mV, respectively), but it makes that of FAD some 40 mV less negative (approximately -340 mV for the beta subunit and -300 mV for FAD bound to the holoenzyme). Binding of the nonreducible NADP(+) analogue, 3-aminopyridine adenine dinucleotide phosphate, made the measured midpoint potential value of the FAD cofactor approximately 30-40 mV less negative in the isolated beta subunit, but had no effect on the redox properties of the alphabeta holoenzyme. This result correlates with the formation of a stable charge-transfer complex between the reduced flavin and the oxidized pyridine nucleotide in the isolated beta subunit, but not in the alphabeta holoenzyme. Binding of L-methionine sulfone, a glutamine analogue, had no significant effect on the redox properties of the enzyme cofactors. On the contrary, 2-oxoglutarate made the measured midpoint potential value of the 3Fe-4S cluster approximately 20 mV more negative in the isolated alpha subunit, but up to 100 mV less negative in the alphabeta holoenzyme as compared to the values of the corresponding free enzyme forms. These findings are consistent with electron transfer from the entry site (FAD) to the exit site (FMN) through the 3Fe-4S center of the enzyme and the involvement of at least one of the two low-potential 4Fe-4S centers, which are present in the glutamate synthase holoenzyme, but not in the isolated subunits. Furthermore, the data demonstrate a specific role of 2-oxoglutarate in promoting electron transfer from FAD to the 3Fe-4S cluster of the glutamate synthase holoenzyme. The modulatory role of 2-oxoglutarate is indeed consistent with the recently determined three-dimensional structure of the glutamate synthase alpha subunit, in which several polypeptide stretches are suitably positioned to mediate communication between substrate binding sites and the enzyme redox centers (FMN and the 3Fe-4S cluster) to tightly control and coordinate the individual reaction steps [Binda, C., et al. (2000) Structure 8, 1299-1308].

Azospirillum brasilense↗
PubMed1999

pH-dependent spectroscopic changes associated with the hydroquinone of FMN in flavodoxins.

Photoreduction with a 5-deazaflavin as the catalyst was used to convert flavodoxins from Desulfovibrio vulgaris, Megasphaera elsdenii, Anabaena PCC 7119, and Azotobacter vinelandii to their hydroquinone forms. The optical spectra of the fully reduced flavodoxins were found to vary with pH in the pH range of 5.0-8.5. The changes correspond to apparent pKa values of 6.5 and 5.8 for flavodoxins from D. vulgaris and M. elsdenii, respectively, values that are similar to the apparent pKa values reported earlier from the effects of pH on the redox potential for the semiquinone-hydroquinone couples of these two proteins (7 and 5.8, respectively). The changes in the spectra resemble those occurring with the free two-electron-reduced flavin for which the pKa is 6.7, but they are red-shifted compared with those of the free flavin. The optical changes occurring with flavodoxins from D. vulgaris and A. vinelandii flavodoxins are larger than those of free reduced FMN. The absorbance of the free and bound flavin increases in the region of 370-390 nm (Delta epsilon = 1-1.8 mM-1 cm-1) with increases of pH. Qualitatively similar pH-dependent changes occur when FMN in D. vulgaris flavodoxin is replaced by iso-FMN, and in the following mutants of D. vulgaris flavodoxin in which the residues mutated are close to the isoalloxazine of the bound flavin: D95A, D95E, D95A/D127A, W60A, Y98S, W60M/Y98W, S96R, and G61A. The 13C NMR spectrum of reduced D. vulgaris [2,4a-13C2]FMN flavodoxin shows two peaks. The peak due to C(4a) is unaffected by pH, but the peak due to C(2) broadens with decreasing pH; the apparent pKa for the change is 6.2. It is concluded that a decrease in pH induces a change in the electronic structure of the reduced flavin due to a change in the ionization state of the flavin, a change in the polarization of the flavin environment, a change in the hydrogen-bonding network around the flavin, and/or possibly a change in the bend along the N(5)-N(10) axis of the flavin. A change in the ionization state of the flavin is the simplest explanation, with the site of protonation differing from that of free FMNH-. The pH effect is unlikely to result from protonation of D95 or D127, the negatively charged amino acids closest to the flavin of D. vulgaris flavodoxin, because the optical changes observed with alanine mutants at these positions are similar to those occurring with the wild-type protein.

Apoproteins↗