Multiple forms of ferredoxin-nicotinamide adenine dinucleotide phosphate reductase from spinach.

Crude extracts of ferredoxin-NADP reductase prepared from spinach by three different methods consistently contained two molecular weight forms of the enzyme: P-1, 117,500, and P-2, 50,000. The lower molecular weight form was purified and shown to consist of two different ionic forms. These three forms of the flavoprotein are immunologically identical. A third molecular weight form of the reductase, excluded by Sephadex G-100, generated P-1 and P-2 on rechromatography. Other experiments demonstrated that this enzyme has NADPH-tetrazolium reductase activity and it accounts for essentially all of the tetrazolium reductase activity of isolated chloroplasts.     

Purification and characterization of ferredoxin-nicotinamide adenine dinucleotide phosphate reductase from a nitrogen-fixing bacterium

Evidence suggesting that Bacillus polymyxa has an active ferredoxin-NADP(+) reductase (EC 1.6.99.4) was obtained when NADPH was found to provide reducing power for the nitrogenase of this organism; direct evidence was provided when it was shown that B. polymyxa extracts could substitute for the native ferredoxin-NADP(+) reductase in the photochemical reduction of NADP(+) by blue-green algal particles. The ferredoxin-NADP(+) reductase was purified about 80-fold by a combination of high-speed centrifugation, ammonium sulfate fractionation, and chromatography on Sephadex G-100 and diethylaminoethyl-cellulose. The molecular weight was estimated by gel filtration to be 60,000. A small amount of the enzyme was further purified by polyacrylamide gel electrophoresis and shown to be a flavoprotein. The reductase was specific for NADPH in the ferredoxin-dependent reduction of cytochrome c and methyl viologen diaphorase reactions; furthermore, NADP(+) was the acceptor of preference when the electron donor was photoreduced ferredoxin. The reductase also has an irreversible NADPH-NAD(+) transhydrogenase (reduced-NADP:NAD oxidoreductase, EC 1.6.1.1) activity, the rate of which was proportional to the concentration of NAD (K(m) = 5.0 x 10(-3)M). The reductase catalyzed electron transfer from NADPH not only to B. polymyxa ferredoxin but also to the ferredoxins of Clostridium pasteurianum, Azotobacter vinelandii, and spinach chloroplasts, although less effectively. Rubredoxin from Clostridium acidi-urici and azotoflavin from A. vinelandii also accept electrons from the B. polymyxa reductase. The pH optima for the various reactions catalyzed by the B. polymyxa ferredoxin-NADP reductase are similar to those of the chloroplast reductase. NAD and acetyl-coenzyme A, which obligatorily activate NADPH- and NADH-ferredoxin reductases, respectively, in Clostridium kluyveri, have no effect on B. polymyxa reductase.     

Role of the C-terminal tyrosine of ferredoxin-nicotinamide adenine dinucleotide phosphate reductase in the electron transfer processes with its protein partners ferredoxin and flavodoxin

The catalytic mechanism proposed for ferredoxin-NADP(+) reductase (FNR) is initiated by reduction of its flavin adenine dinucleotide (FAD) cofactor by the obligatory one-electron carriers ferredoxin (Fd) or flavodoxin (Fld) in the presence of oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)). The C-terminal tyrosine of FNR, which stacks onto its flavin ring, modulates the enzyme affinity for NADP(+)/H, being removed from this stacking position during turnover to allow productive docking of the nicotinamide and hydride transfer. Due to its location at the substrate-binding site, this residue might also affect electron transfer between FNR and its protein partners. We therefore studied the interactions and electron-transfer properties of FNR proteins mutated at their C-termini. The results obtained with the homologous reductases from pea and Anabaena PCC7119 indicate that interactions with Fd or Fld are hardly affected by replacement of this tyrosine by tryptophan, phenylalanine, or serine. In contrast, electron exchange is impaired in all mutants, especially in the nonconservative substitutions, without major differences between the eukaryotic and the bacterial FNR. Introduction of a serine residue shifts the flavin reduction potential to less negative values, whereas semiquinone stabilization is severely hampered, introducing further constraints to the one-electron-transfer processes. Thus, the C-terminal tyrosine of FNR plays distinct and complementary roles during the catalytic cycle, (i) by lowering the affinity for NADP(+)/H to levels compatible with steady-state turnover, (ii) by contributing to the flavin semiquinone stabilization required for electron splitting, and (iii) by modulating the rates of electron exchange with the protein partners.     

Ethylisocyanide equilibria of hemoglobins M Iwate, M Boston, M Hyde Park, M Saskatoon, and M Milwaukee-I in half-ferric and fully reduced states.

The ethylisocyanide equilibria of all the five known hemoglobins M, namely Hb M Iwate (alpha287 Tyrbeta2), Hb M Boston (alpha258 Tyrbeta2), Hb M Hyde Park (alpha2beta292 Tyr), Hb M Saskatoon (alpha2beta263 tyr), and Hb M Milwaukee-I (alpha2beta267 Glu), were studied both in the half-ferric and fully reduced heme states. In the half-ferric state, no heme-heme interaction was observed for Hb M Iwate, Hb M Boston, and Hb M Hyde Park, but Hb M Saskatoon and Hb M Milwaukee-I show small but definite heme-heme interaction with Hill's n of 1.3. The beta chain mutants, Hb M Hyde Park and Hb M Saskatoon, have almost normal affinity for ethylisocyanide and a normal Bohr effect, whereas the alpha chain mutants, Hb M Iwate and Hb M Boston, have abnormally low affinity and almost no Bohr effect. Hb M Milwaukee-I showed a large Bohr effect and low affinity. These results are consistent qualitatively with those on oxygen equilibria reported previously. In the fully reduced state, in which all four hemes were in the ferrous state and capable of binding ethylisocyanide distinct differences were found in the extent of heme-heme interaction. Namely, the n values for proximal histidine mutants, Hb M Iwate and Hb M Hyde Park, were 1.1 and 1.0, respectively, whereas the distal histidine mutants, Hb M Boston and Hb M Saskatoon, showed high n values of 2.4 and 1.6, respectively. Hb M Milwaukee-I also exhibited a high n value of 2.0 The ethylisocyanide affinity of the four histidine mutants was high compared with that of Hb A, while that for Hb M Milwaukee-I was almost normal. All five Hbs M had approximately normal magnitudes of Bohr effect. In the half-ferric state, the proximal and distal histidine mutants of the same chain showed similar affinity for ethylisocyanide and Bohr effect, rather different from those of the mutants of the opposite chain. These differences seem to be derived from the difference of abnormal bonding of ferric iron to tyrosine or glutamic acid. On the other hand, the reduction of iron, which abolished the abnormal bonding and made all of the chains capable of binding ligand, extinguished the differences of alpha and beta chains, and the effect of amino acid side chains close to iron on ligand binding properties became clear. Proximal histidine, which is considered to trigger the transition between the T and R states, seems to be essential to the heme-heme interaction.     

Selective inactivation of NADPH-cytochrome P-450 (c) reductase by diazotized 3-aminopyridine adenine dinucleotide phosphate

Cyanogen-bromide cleaved glucagon has been extensively purified in yields of 80–85% by the use of gel filtration and by cation-exchange chromatography at pH 4.5–5.2. This pH range maintains a charge difference between the holohormone and its cleavage product, the truncated homoserine lactone derivative, yet maintains the integrity of the lactone ring. Purity is determined by the lack of methionine and the presence of homoserine following peptide hydrolysis. The homoserine lactone is opened by treatment with 0.2 n triethylamine at pH 9.5. The lactone can be reformed by treatment with trifluoroacetic acid for 1 h at room temperature although protection against photooxidation of tryptophan-25 must be provided. The homoserine lactone form binds less well to glucagon receptors than does the homoserine form. Adenylate cyclase is activated by the lactone to an extent comparable to that obtained by native hormone but at elevated concentrations. The procedures described may be useful for purification of other cyanogen bromide cleavage products and is useful for semisynthetic methods based upon cyanogen bromide-cleaved derivatives of glucagon.     

Kinetic isotope effects on the oxidation of reduced nicotinamide adenine dinucleotide phosphate by the flavoprotein methylenetetrahydrofolate reductase

Previous work from this laboratory has established that the NADPH-menadione oxidoreductase reaction catalyzed by methylenetetrahydrofolate reductase from pig liver proceeds by Ping Pong Bi Bi kinetics and that the reductive half-reaction is rate limiting in steady-state turnover. We have now shown that methylenetetrahydrofolate reductase stereo-specifically removes the pro-S hydrogen from the 4-position of NADPH. During the oxidation of [4(S)-3H]NADPH, we observed a kinetic isotope on V/KNADPH of 10.8 +/- 0.4. When comparing the rates of oxidation of [4(S)-2H]NADPH and [4(S)-1H]NADPH, we measure kinetic isotope effects on V of 4.78 +/- 0.15 and on V/KNADPH of 4.54 +/- 0.59. When oxidation of [4(R)-2H]NADPH and [4(R)-1H]NADPH is compared, the secondary kinetic isotope effect on V is 1.04 +/- 0.01. When the NADPH-menadione oxidoreductase reaction is catalyzed in tritiated water, no incorporation of solvent tritium into residual NADPH is observed. We conclude from these observations that the oxidation of NADPH is largely or entirely rate limiting in the reductive half-reaction and, hence, in NADPH-menadione oxidoreductase turnover at saturating menadione concentration. In the presence of saturating NADPH, the flavin reduction proceeds with a rate constant of 160 S-1, which is at least 29-fold slower than estimates of the lower limit for the diffusion-limited rate constant characterizing NADPH binding to the enzyme under physiological conditions. Albery & Knowles have defined criteria for perfection in enzyme catalysis [Albery, W. J., & Knowles, J.R. (1976) Biochemistry 15, 5631-5640].(ABSTRACT TRUNCATED AT 250 WORDS)