The thermodynamics of flavin binding to the apoflavodoxin from Azotobacter vinelandii

A thermodynamic study of the binding of flavins (FMN, FAD, 8-carboxylic acid-riboflavin) to the purified apoflavodoxin from Azotobacter vinelandii has been conducted. The binding of FMN was studied at a number of temperatures (10,15, 20, 25, and 30 °C), pH's (6.0, 7.4, and 9.0), and buffer conditions. The binding of FAD was studied at pH 7.4 and 25 °C under a number of buffer conditions. The binding of 8-carboxylic acid-riboflavin to the apoflavodoxin and the binding of FMN to the dimeric form of the apoflavodoxin were investigated at pH 7.4 and 25 °C. Enthalpies of binding for FMN, FAD, and 8-carboxylic- acid-riboflavin were ?28.3, ?16.6, and ?14.0 kcal mol^?1, respectively. The enthalpy of binding of FMN to the dimeric form of the apoflavodoxin was ?22.2 kcal mol of binding sites^?1. Binding constants of about 10⁸,10⁶, and 10⁶ were obtained for the binding of FMN, FAD, and 8-carboxylic acid-riboflavin, respectively. Using established thermodynamic relationships free energy and entropy changes were calculated. The entropy data indicate that a large degree of ordering of the system occurs upon flavin binding. The pH data suggest that FMN may bind in both the mono-and dianion forms, and that binding doesn't change the pK_a of any functional group in the system. It appears that the phosphate group is probably responsible for approximately half the binding enthalpy observed for the binding of FMN. The temperature-dependence data over the temperature range studied is biphasic, centered at 20 °C, indicating that flavin binding occurs to the protein in two thermodynamic states corresponding to the two heat capacities observed. These findings are used to discuss a model for flavin binding.     

Last in, first out: the role of cofactor binding in flavodoxin folding

Although many proteins require the binding of a ligand to be functional, the role of ligand binding during folding is scarcely investigated. Here, we have reported the influence of the flavin mononucleotide (FMN) cofactor on the global stability and folding kinetics of Azotobacter vinelandii holoflavodoxin. Earlier studies have revealed that A. vinelandii apoflavodoxin kinetically folds according to the four-state mechanism: I(1) <=> unfolded apoflavodoxin <=> I(2) <=> native apoflavodoxin. I(1)an off-pathway molten globule-like is intermediate that populates during denaturant-induced equilibrium unfolding; I(2) is a high energy on-pathway folding intermediate that never populates to a significant extent. Here, we have presented extensive denaturant-induced equilibrium unfolding data of holoflavodoxin, holoflavodoxin with excess FMN, and apoflavodoxin as well as kinetic folding and unfolding data of holoflavodoxin. All folding data are excellently described by a five-state mechanism: I(1) + FMN <=> unfolded apoflavodoxin + FMN <=> I(2) + FMN <=> native apoflavodoxin + FMN<=> holoflavodoxin. The last step in flavodoxin folding is thus the binding of FMN to native apoflavodoxin. I(1),I(2), and unfolded apoflavodoxin do not interact to a significantextent with FMN. The autonomous formation of native apoflavodoxin is essential during holoflavodoxin folding. Excess FMN does not accelerate holoflavodoxin folding, and FMN does not act as a nucleation site for folding. The stability of holoflavodoxin is so high that even under strongly denaturing conditions FMN needs to be released first before global unfolding of the protein can occur.     

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.     

A thermodynamic description of the self-association of flavin mononucleotide.

Systematic heat of dilution studies of the self-association of flavin mononucleotide (FMN) have been conducted as a function of ionic strength (0.05 – 2.0 m) and pH (5–9) in aqueous solution. The data are adequately described by the expression $\text{Q}{}_{\mathrm{T}}\text{= ΔH ? (}\text{ΔH}\text{K}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(}\text{Q}{}_{\mathrm{T}}\text{c}{}_{\mathrm{T}}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for an isodesmic self-association. Q_T is the molar heat of dilution, ΔH and K are the derived enthalpy and equilibrium constants for the process FMN + (FMN)_i?1 ? (FMN)_i, and c_T is the concentration of FMN expressed in monomer units. Typical values derived for the various thermodynamic parameters at 25 °C are ΔG = ?3.56 kcal mol^?1, ΔH = ?3.72 kcal mol^?1, and ΔS = ?0.54 cal (mol · deg)^?1. These data, plus nuclear magnetic resonance evidence (Yagi, K., Ohishi, N., Takai, A., Kawano, K., and Kyogoku, Y., 1976, Biochemistry15, 2877–2880) argue in favor of an open-ended association of flavin molecules. The signs of the various thermodynamic parameters suggest that both hydrophobic and surface energy forces contribute significantly to the association, while the lack of any significant ionic strength dependence indicates the lack of any ionic centers in the association.     

The reduced nicotinamide adenine dinucleotide “oxidase” of Acholeplasma laidlawii membranes

An NADH dehydrogenase possessing a specific activity 3–5 times that of membrane-bound enzyme was obtained by extraction of Acholeplasma laidlawii membranes with 9.0 % ethanol at 43 °C. This dehydrogenase contained only trace amounts of iron (suggesting an uncoupled respiration), a flavin ratio of 1 : 2 FAD to FMN, and 30–40 % lipid. Its resistance to sedimentation is probably due to the high flotation density of the lipids. It efficiently utilized ferricyanide, menadione and dichlorophenol indophenol as electron acceptors, but not O₂, ubiquinone $\text{Q}{}_{10}$ or cytochrome $\text{c}$. Lineweaver-Burk plots of the dehydrogenase were altered to linear functions upon extraction with 9.0 % ethanol. A secondary site of ferricyanide reduction could not be explained by the presence of cytochromes, which these membranes lack. In comparison to other respiratory chain-linked NADH dehydrogenases in cytochrome-containing respiratory chains, this dehydrogenase was characterized by similar $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{'}\text{s}$ with ferricyanide, dichlorophenol indophenol, menadione as electron acceptors, but considerably smaller $\text{V'}\text{s}$ with ferricyanide, dichlorophenol indophenol, menadione as electron acceptors, and smaller specific activities. It was not stimulated or reactivated by the addition of FAD, FMN, Mg²⁺, cysteine or membrane lipids, and was less sensitive to respiratory inhibitors than unextracted enzyme. The ineffectiveness of ADP stimulation on O₂ uptake, the insensitivity to oligomycin and the very low iron content of A. laidlawii membranes were considered in relation to conservation of energy by these cells. Some kinetic properties of the dehydrogenation, the uniquely high glycolipid content and apparently uncoupled respiration at Site I were noteworthy characteristics of this NADH dehydrogenase from the truncated respiratory chain of A. laidlawii.     

Effect of uncouplers on anaerobic adaptation of hydrogenase activity in C., reinhardtii

An anaerobic incubation period of varying duration is required to induce hydrogenase activity in $\text{C.}\text{,}\text{reinhardtii}$. Inclusion of sodium acetate, a metabolizable carbonaceous substrate, in the medium during anaerobic incubation accelerates the activation process. Thus, in the presence of sodium acetate, hydrogen photoproduction is detected within 7 to 15 minutes after the onset of anaerobiosis. On the contrary, if an uncoupler of phosphorylation, such as CCCP or sodium arsenate, is present during anaerobic incubation, little activation of the hydrogenase is observed even after hours of anaerobic adaptation. Since the uncouplers had no inhibitory effect on hydrogen photoproduction by the alga when added to previously activated cells, they are not inhibitors of activated hydrogenase. The uncouplers interfere, most likely, with the activation of hydrogenase. Similar effects of uncouplers on the hydrogenase activation process were obtained using a cell-free assay of hydrogenase activity. These observations provide strong evidence that anaerobic activation of the hydrogenase is an energy requiring process.     

Common conformational changes in flavodoxins induced by FMN and anion binding: the structure of Helicobacter pylori apoflavodoxin

Flavodoxins, noncovalent complexes between apoflavodoxins and flavin mononucleotide (FMN), are useful models to investigate the mechanism of protein/flavin recognition. In this respect, the only available crystal structure of an apoflavodoxin (that from Anabaena) showed a closed isoalloxazine pocket and the presence of a bound phosphate ion, which posed many questions on the recognition mechanism and on the potential physiological role exerted by phosphate ions. To address these issues we report here the X-ray structure of the apoflavodoxin from the pathogen Helicobacter pylori. The protein naturally lacks one of the conserved aromatic residues that close the isoalloxazine pocket in Anabaena, and the structure has been determined in a medium lacking phosphate. In spite of these significant differences, the isoallozaxine pocket in H. pylori apoflavodoxin appears also closed and a chloride ion is bound at a native-like FMN phosphate site. It seems thus that it is a general characteristic of apoflavodoxins to display closed, non-native, isoalloxazine binding sites together with native-like, rather promiscuous, phosphate binding sites that can bear other available small anions present in solution. In this respect, both binding energy hot spots of the apoflavodoxin/FMN complex are initially unavailable to FMN binding and the specific spot for FMN recognition may depend on the dynamics of the two candidate regions. Molecular dynamics simulations show that the isoalloxazine binding loops are intrinsically flexible at physiological temperatures, thus facilitating the intercalation of the cofactor, and that their mobility is modulated by the anion bound at the phosphate site.     

On the active site of hydrogenase from Chromatium vinosum

Isotope substitution of ⁵⁷Fe ($\text{I =}\text{1}\text{2}$) for ⁵⁶Fe has a pronounced effect on the two EPR signals of hydrogenase of Chromatium vinosum. It is proposed that signal 1, the intensity of which is increased several-fold by a deoxygenation-oxygenation cycle with a simultaneous increase of a signal from Fe³⁺, is due to a [3Fe-xS] cluster. It is further proposed that signal 2 is caused by a magnetic interaction of a [4Fe-4S]³⁺ cluster with an unidentified paramagnet. The addition of 10 μM Ni to the culture medium (already containing 1 μM Ni) increased the enzyme activity 3–6-fold, without effect on the growth of the bacterium. Addition of ⁶¹Ni ($\text{I =}\text{3}\text{2}$) to the medium did not change the EPR spectrum of hydrogenase. From a comparison of the EPR signal intensities and the enzyme activities it is concluded that, in the hydrogenase preparation as isolated, molecules containing a [3Fe-xS) cluster are not active, and that active molecules have a [4Fe-4S]^3+(3+,2+) cluster plus an as yet unidentified paramagnetic redox component. The latter is thought to be the primary site of interaction of the enzyme with H₂. Ni is considered as a possible candidate for this component.     

The enthalpy of oxidation of flavin mononucleotide: Temperature dependence of in vitro bacterial luciferase bioluminescence

The enthalpy of the bioluminescent reaction

$\text{FMNH}{}_2\text{+ RCHO + O}{}_2\text{→}\text{luciferase}\text{FMN + RCOO + H}{}_3\text{O}{}^{\mathrm{+}}\text{+ hv}$

has been studied by direct calorimetric methods. Bacterial luciferase, isolated from Beneckea harveyi (formerly strain MAV) has been used to catalyze the oxidation of reduced flavin mononucleotide (FMNH₂) and a long chain aliphatic aldehyde (dodecanal, RCHO) by molecular oxygen to give the indicated products and blue-green light. The enthalpy measured for this process was found to be ΔH_L = ?338.9 k.J (mol FMN)^?1 (?81.0 kcal) at 25.00 °C and ?402.9 kJ (mol FMN)^?1 (?96.3 kcal) at 7.00 °C. Calculations based on redox electrode potentials indicate a corresponding value of the free energy change, ΔG_L = ?464.8 kJ (mol FMN)^?1 (?111.1 kcal), at 25 °C. Measurements were performed in 0.15 m phosphate buffer, pH 7.0 and the values were arrived at by correcting the observed heats for the heat associated with the autoxidation process: FMNH₂ + O₂ ? FMN + H₂O₂; ΔH_D = ?158.5 kJ (mol FMN)^?1 (?37.8). These data and a detailed thermodynamic analysis have demonstrated the need for two parameters, referred to as the intrinsic free energy, ΔG₁, and intrinsic enthalpy, ΔH₁, which are functionally defined by the relations ΔG_I = ΔG_L ? uhvΔH_I = ΔH_L ? uhv, where u is the quantum yield of the reaction expressed in einsteins mole^?1.These parameters reflect the thermochemistry of the bioluminescent reaction corrected for emitted photons. Thus, they are useful for comparing the thermochemistry of a chemiluminescent process. Their values for the bacterial luciferase system at 25 °C and pH 7.0 are ?391.6 and ?266.9 kJ (mol FMN)^?1 (?93.6 and ?63.8 kcal), respectively, assuming a value of 0.3 for the quantum yield. The calorimetric data also suggest the existence of a long-lived species which persists after photon emission.     

Reactivity of an FAD-dependent oxygenase with free flavins: a new mode of uncoupling in flavoprotein oxygenases.

Conversion of 2-methyl-3-hydroxypyridine-5-carboxylic acid (Cpd I) to α-(N-acetylaminomethylene)succinic acid (Cpd A) is catalyzed by an FAD protein, Cpd I oxygenase (Sparrow, et al., J. Biol. Chem. [1969] $\text{244}$, 2590–2600) according to the equation: Cpd I + O₂ + NADH + H⁺ → Cpd A + NAD⁺. When free FAD, FMN or riboflavin is added to reaction mixtures, oxidation of NADH remains dependent on presence of oxygenase and Cpd I, but is partially uncoupled from the oxygenation of Cpd I. Under these conditions, free reduced flavins appear in solution, as shown by their ability to reduce cytochrome c. These effects are not due to an increased rate of NADH oxidation. Such uncoupling may lead to appearance of artifactual species of activated oxygen or flavin that play no intermediate role in the oxygenase reaction.     

Plasma membrane NADH dehydrogenase and Ca2+-dependent potassium transport in erythrocytes of several animal species

Ca²⁺-dependent K⁺ transport and plasma membrane NADH dehydrogenase activities have been studied in several ‘high-K⁺’ (human, rabbit and guinea pig) and ‘low-K⁺’ (dog, cat and sheep) erythrocytes. All the species except sheep showed Ca²⁺-dependent K⁺ transport. NADH-ferricyanide reductase was detected in all the species and showed positive correlation with the flavin contents of the membranes. NADH-cytochrome $\text{c}$ reductase was very low or absent in dog, sheep and guinea pig membranes. No correlation was found between NADH dehydrogenase and Ca²⁺-dependent K⁺ channel activities in the species studied. Nor were any of the above activities correlated with $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity.     

Phosphates of riboflavin and riboflavin analogs: A reinvestigation by high-performance liquid chromatography

Phosphoric acid esters of riboflavin can be easily separated by reverse-phase high-performance liquid chromatography using eluants of 0.1 M ammonium formate in aqueous methanol. Commercial FMN preparations contained seven different flavin phosphates; the content of riboflavin 5'-phosphate was 70-75% and is in agreement with previous studies. Millimole amounts of crude FMN can be processed by preparative HPLC. The method permits the preparation of greater than 99%-pure 5'-FMN. The following compounds were isolated in pure form and their structures determined: riboflavin 4'-phosphate, riboflavin 3'-phosphate, riboflavin 4',5'-diphosphate; riboflavin 3',4'-diphosphate, and riboflavin 3',5'-diphosphate. The latter compound binds tightly to apoflavodoxin from Megasphaera elsdenii (KD = 9.7 X 10(-9) M). The bound flavin has high catalytic activity, thus representing a novel type of FMN analog. A wide variety of structural analogs of FMN can be obtained in pure form by preparative HPLC.     

Mechanism of flavin mononucleotide cofactor binding to the Desulfovibrio vulgaris flavodoxin. 1. Kinetic evidence for cooperative effects associated with the binding of inorganic phosphate and the 5'-phosphate moiety of the cofactor

The pathway(s) by which the flavin cofactor binds to the apoflavoprotein is the subject of some debate. The crystal and NMR structures of several different flavodoxins have provided some insight, although there is disagreement about the location of the initial interaction between the flavin mononucleotide (FMN) and the apoflavodoxin and the degree of protein conformational change associated with cofactor binding [Genzor, C. G., Perales-Alcon, A., Sancho, J., and Romero, A. (1996) Nat. Struct. Biol. 3, 329-332; Steensma, E., and van Mierlo, C. P. M. (1998) J. Mol. Biol. 282, 653-666]. Binding kinetics using stopped-flow spectrofluorimetry and phosphate competition studies were used to develop a model for flavin binding to the flavodoxin from Desulfovibrio vulgaris. In the presence of phosphate, the time course of fluorescence quenching associated with FMN binding to apoflavodoxin was biphasic, whereas riboflavin, which lacks the 5'-phosphate group of FMN, displayed monophasic binding kinetics. When the concentration of phosphate in solution was increased, the FMN binding rates of the two phases behaved differently; the rate of one phase decreased, while the rate of the other increased. A similar increase in the single phase associated with riboflavin binding was also observed. This has led to the following model. The binding of the flavin isoalloxazine ring to its subsite is dependent on the presence of a phosphate group in the phosphate-binding subsite. When phosphate is in the buffer solution, FMN can bind in either of two ways: by the initial insertion of the 5'-phosphate group followed by ring binding or, when inorganic phosphate from solution is bound, the insertion of the isoalloxazine ring first. Riboflavin, which lacks the phosphate moiety of FMN, binds only in the presence of inorganic phosphate, presumably due to the binding of this group in the phosphate-binding subsite. These results suggest that cooperative interactions exist between the phosphate subsite and the ring-binding region in the D. vulgaris flavodoxin that are necessary for isoalloxazine ring binding.     

Magnetic interaction of nickel(III) and the iron-sulphur cluster in hydrogenase from Chromatium vinosum

Upon partial reduction of hydrogenase from Chromatium vinosum with ascorbate plus phenazine methosulphate, EPR signals due to Ni(III) and a [3Fe-xS] cluster appear simultaneously and with equal intensities. Since the intact enzyme shows no $\text{S =}\text{1}\text{2}$ signals, it is concluded that Ni(III) and a [4Fe-4S]³⁺ cluster interact magnetically in such a way as to prevent the detection of the two paramagnets as individual $\text{S =}\text{1}\text{2}$ systems. This interaction is thought to be the origin of a signal in which Fe is involved and which is not due to an $\text{S =}\text{1}\text{2}$ system (Albracht, S.P.J., Albrecht-Ellmer, K.J., Schmedding, D.J.M. and Slater, E.C. (1982) Biochim. Biophys. Acta 681, 330–334). A variable fraction of the enzyme preparation shows signals due to Ni(III) and a [3Fe-xS] cluster with equal intensities without any further treatment. These are thought to be derived from irreversibly inactivated enzyme molecules. The enzyme contains no selenium.     

Studies on the biosynthesis of cytochrome P-450 in rat liver--a probe with phenobarbital.

The reaction of NAD(P)H:flavin oxidoreductase (flavin reductase) from Photobacterium fischeri is proposed to follow a ping-pong bisubstrate-biproduct mechanism. This is based on a steady-state kinetic analysis of initial velocities and patterns of inhibition by NAD⁺ and AMP. The double reciprocal plots of initial velocities versus concentrations of FMN or NADH show, in both cases, a series of parallel lines. The Michaelis constants for NADH (FMN saturating) and FMN (NADH saturating) are 2.2 and 1.2 × 10^?4m, respectively. The product NAD⁺ has been found to be an inhibitor competitive with FMN but non-competitive with NADH. Using AMP as an inhibitor, noncompetitive inhibition patterns were observed with respect to both NADH and FMN as the varied substrate. In addition, the reductase was not inactivated by treatment with N-ethylmaleimide either alone or in the presence of FMN, but the enzyme was inactivated by N-ethylmaleimide in the presence of NADH. These findings suggest that flavin reductase shuttles between disulfide- and sulfhydryl-containing forms during catalysis.     

H2 metabolism in photosynthetic organisms. II. Light-dependent H2 evolution by preparations from Chlamydomonas, Scenedesmus and spinach.

Light-dependent H₂ evolution from dithiothreitol as electron donor was observed with cell-free preparations of anaerobically adapted $\text{Chlamydomonas reinhardii, Scenedesmus obliquus}$ and from spinach chloroplasts mixed with $\text{Chlamydomonas}$ hydrogenase. NADH substituted for dithiothreitol as electron donor only in the $\text{Chlarmydomonas}$ preparation. Dibromothymoquinone, an antagonist of plastoquinone, selectively inhibited H₂ photoevolution from NADH. These results are interpreted as indicating that 3-(3,4-dichlorophenyl)-1,1-dimethyl urea insensitive H₂ photoevolution by algae containing hydrogenase is due to the capability of NADH to reduce plastoquinone in the electron transport chain, and to evolve H₂ by a low redox potential carrier of photosystem I.     

Separation of the apoprotein and reconstitution of the holoprotein from the long-lived intermediate in bacterial bioluminescence

A long-lived intermediate in bacterial bioluminescence, which has been suggested to be an FMN flavoprotein, has been separated as an apoprotein plus free FMN and the holoprotein reconstituted by addition of FMN (K_a = 7 × 10⁵ M^?1). The apoprotein preparation reacts with long-chain aldehyde to give the full quantum yield observed for the complete system. Only after removal of all remaining FMN in the apoprotein preparation by prior dialysis of luciferase against KBr and inclusion of apoflavodoxin in the reaction mixture, can a dependence of the light output on FMN be observed. Bacterial bioluminescence therefore appears to be in the class of sensitized chemiluminescence with FMN acting as the specific sensitizing agent.     

Replacement of sulfide by selenide in the [4Fe-4S] clusters of the ferredoxin fromClostridium pasteurianum

The sulfur atoms of the two [4Fe-4S] clusters present in the ferredoxin from $\text{C. pasteurianum}$ have been replaced by selenium. The optical absorption spectrum of the Se-ferredoxin is slightly different from the spectrum of the native protein, but it displays the characteristic features of [4Fe-4X] ( X = S, Se) clustors. The reduced Se-ferredoxin can reduce hydrogenase, and the oxidized Se-ferredoxin can be reduced by hydrogenase in the presence of molecular hydrogen. This is the first report of sulfide substitution by selenide in an iron-sulfur protein containing [4Fe-4S] active sites.     

Understanding the FMN cofactor chemistry within the Anabaena Flavodoxin environment

The chemical versatility of flavin cofactors within the flavoprotein environment allows them to play main roles in the bioenergetics of all type of organisms, particularly in energy transformation processes such as photosynthesis or oxidative phosphorylation. Despite the large diversity of properties shown by flavoproteins and of the biological processes in which they are involved, only two flavin cofactors, FMN and FAD (both derived from the 7,8-dimethyl-10-(1′-D-ribityl)-isoalloxazine), are usually found in these proteins. Using theoretical and experimental approaches we have carried out an evaluation of the effects introduced upon substituting the 7- and/or 8-methyls of the isoalloxazine ring in the chemical and oxido-reduction properties of the different atoms of the ring on free flavins and on the photosynthetic Anabaena Flavodoxin (a flavoprotein that replaces Ferredoxin as electron carrier from Photosystem I to Ferredoxin-NADP⁺ reductase). In Anabaena Flavodoxin both the protein environment and the redox state contribute to modulate the chemical reactivity of the isoalloxazine ring. Anabaena apoflavodoxin is shown to be designed to stabilise/destabilise each one of the FMN redox states (but not of the analogues produced upon substitution of the 7- and/or 8-methyls groups) in the adequate proportions to provide Flavodoxin with the particular properties required for the functions in which it is involved in vivo. The 7- and/or 8-methyl groups of the ixoalloxazine can be discarded as the gate for electrons exchange in Anabaena Fld, but a key role in this process is envisaged for the C6 atom of the flavin and the backbone atoms of Asn58.     

The presence of redox-sensitive nickel in the periplasmic hydrogenase from Desulfovibrio gigas

A new and improved method for the purification of the periplasmic hydrogenase from $\text{Desulfovibrio}\text{gigas}$is described. This preparation of hydrogenase was found to contain 0.64 g atom of nickel per mole of protein. In the oxidized state, the hydrogenase exhibited an isotropic signal at g = 2.02 and a characteristic Ni(III) signal with g-values at 2.31, 2.20 and ～2.0. The EPR spectrum of the reduced enzyme consisted of multiple species. One set of g-values are determined as 2.17, 2.08 and 2.04. The other minor species exhibited a resonance at g = 2.28. On partial reoxidation of the hydrogenase, the initial Ni(III) signals reappeared along with additional signals attributed to multiple Ni(III) species. It is proposed that Ni is an important functional unit in this hydrogenase.