Biomedical subjects

D B Knaff

Publications and source records attributed to D B Knaff.

At least 37 records · Page 2Linked to original sources

PubMed1998

Oxidation-reduction properties of the regulatory site of spinach phosphoribulokinase.

The oxidation-reduction midpoint potential (Em) of the regulatory disulfide, formed between Cys16 and Cys55, of spinach chloroplast phosphoribulokinase has been determined both for the wild-type enzyme and for a C244S-C250S double mutant, using enzymatic activity to monitor the oxidation-reduction state of the regulatory disulfide. At pH 7.0, Em values for the two-electron reduction of the regulatory disulfide of -295 +/- 10 and -290 +/- 10 mV were measured for the wild-type and mutant, respectively. In contrast to the dependence of activity on ambient potential (Eh) observed for the wild-type enzyme and the double mutant, which both followed the Nernst equation for a two-electron process, high and constant activity was exhibited by a C16S-C244S-C250 triple mutant of the enzyme at all Eh values tested. Em values for the wild-type enzyme were also measured at pH values of 6.7, 7.5, 7.7, and 8.2 and the Em vs pH data in this region give a good fit to a straight line with a slope of -60 mV/pH unit.

Chloroplasts↗

PubMed1997

The ferredoxin-binding site of ferredoxin: Nitrite oxidoreductase. Differential chemical modification of the free enzyme and its complex with ferredoxin.

Spinach (Spinacea oleracea) leaf ferredoxin (Fd)-dependent nitrite reductase was treated with either the arginine-modifying reagent phenyl-glyoxal or the lysine-modifying reagent pyridoxal-5'-phosphate under conditions where only the Fd-binding affinity of the enzyme was affected and where complex formation between Fd and the enzyme prevented the inhibition by either reagent. Modification with [14C]phenylglyoxal allowed the identification of two nitrite reductase arginines, R375 and R556, that are protected by Fd against labeling. Modification of nitrite reductase with pyridoxal-5'-phosphate, followed by reduction with NaBH4, allowed the identification of a lysine, K436, that is protected by Fd against labeling. Positive charges are present at these positions in all of the Fd-dependent nitrite reductase for which sequences are available, suggesting that these amino acids are directly involved in electrostatic binding of Fd to the enzyme.

Amino Acid Sequence↗

PubMed1996

Isolation and characterization of vibrational spectra of individual heme active sites in cytochrome bc1 complexes from Rhodobacter capsulatus.

Resonance Raman spectra of bc1 complexes and isolated c1 subunit from Rhodobacter capsulatus have been obtained using a variety of excitation wavelengths. Spectra obtained via Q-band excitation of bc1 complexes in different redox states were separated to yield the individual vibrational spectra of each of the three heme active sites. Hemes bH and c1 exhibit vibrational spectra typical of b- and c-type hemes, respectively. In contrast, the spectrum of heme bL is anomalous with respect to those of other hemes b. The isolated spectra were also used to assess the effects of inhibitor binding on the local structural environments of the hemes. Neither antimycin nor myxothiazol binding produces dramatic structural perturbations at the hemes. Heme c1 is completely unaffected by the presence of either inhibitor. The vibrational spectra of hemes bH and bL are slightly altered by antimycin and myxothiazol binding, respectively.

Antimycin A↗

PubMed1996

The function and properties of the iron-sulfur center in spinach ferredoxin: thioredoxin reductase: a new biological role for iron-sulfur clusters.

Thioredoxin reduction in chloroplasts is catalyzed by a unique class of disulfide reductases which use a [2Fe-2S]2+/+ ferredoxin as the electron donor and contain an Fe-S cluster as the sole prosthetic group in addition to the active-site disulfide. The nature, properties, and function of the Fe-S cluster in spinach ferredoxin:thioredoxin reductase (FTR) have been investigated by the combination of UV/visible absorption, variable-temperature magnetic circular dichroism (MCD), EPR, and resonance Raman (RR) spectroscopies. The results indicate the presence of an S = 0 [4Fe-4S]2+ cluster with complete cysteinyl-S coordination that cannot be reduced at potentials down to -650 mV, but can be oxidized by ferricyanide to an S = 1/2 [4Fe-4S]3+ state (g = 2.09, 2.04, 2.02). The midpoint potential for the [4Fe-4S]3+/2+ couple is estimated to be +420 mV (versus NHE). These results argue against a role for the cluster in mediating electron transport from ferredoxin (Em = -420 mV) to the active-site disulfide (Em = -230 mV, n = 2). An alternative role for the cluster in stabilizing the one-electron-reduced intermediate is suggested by parallel spectroscopic studies of a modified form of the enzyme in which one of the cysteines of the active-site dithiol has been alkylated with N-ethylmaleimide (NEM). NEM-modified FTR is paramagnetic as prepared and exhibits a slow relaxing, S = 1/2 EPR signal, g = 2.11, 2.00, 1.98, that is observable without significant broadening up to 150 K. While the relaxation properties are characteristic of a radical species, MCD, RR, and absorption studies indicate at least partial cluster oxidation to the [4Fe-4S]3+ state. Dye-mediated EPR redox titrations indicate a midpoint potential of -210 mV for the one-electron reduction to a diamagnetic state. By analogy with the properties of the ferricyanide-oxidized [4Fe-4S] cluster in Azotobacter vinelandii 7Fe ferredoxin [Hu, Z., Jollie, D., Burgess, B. K., Stephens, P. J., & Münck, E. (1994) Biochemistry 33, 14475-14485], the spectroscopic and redox properties of NEM-modified FTR are interpreted in terms of a [4Fe-4S]2+ cluster covalently attached through a cluster sulfide to a cysteine-based thiyl radical formed on one of the active-site thiols. A mechanistic scheme for FTR is proposed with similarities to that established for the well-characterized NAD(P)H-dependent flavin-containing disulfide oxidoreductases, but involving sequential one-electron redox processes with the role of the [4Fe-4S]2+ cluster being to stabilize the thiyl radical formed by the initial one-electron reduction of the active-site disulfide. The results indicate a new biological role for Fe-S clusters involving both the stabilization of a thiyl radical intermediate and cluster site-specific chemistry involving a bridging sulfide.

Chloroplasts↗

PubMed1996

Isolation and characterization of soluble electron transfer proteins from Chromatium purpuratum.

Several soluble electron transfer proteins were isolated and characterized from the marine purple-sulfur bacterium Chromatium purpuratum. The C. purpuratum flavocytochrome c is similar in molecular mass (68 kDa) and isoelectric point (6.5) to flavocytochromes isolated from other phototrophs. Redox titrations of the flavocytochrome c hemes show two components with midpoint potential values of +15 and -120 mV, behavior similar to that observed with the flavocytochrome isolated from the thermophilic Chromatium tepidum. Moreover, N-terminal amino acid sequence analysis of both the flavin and the cytochrome subunit indicates substantial homology to the primary structure of the flavocytochrome c of Chromatium vinosum. In contrast, the C. purpuratum high-potential iron-sulfur protein (HiPIP) differs from those isolated from other photosynthetic bacteria in its relatively high midpoint potential (+390 mV) and the possibility that it exists as a dimer in solution. Two low molecular mass c-type cytochromes were also characterized. One appears to be a high-potential (+310 mV) c8-type cytochrome. Amino acid sequencing suggests that the second cytochrome may be a homologue of the low-potential cytochrome c-551, previously described in two species of Ectothiorhodospirillaceae.

Amino Acid Sequence↗

PubMed1996

Oxidation-reduction and transient kinetic studies of spinach ferredoxin-dependent glutamate synthase.

Spinach leaf ferredoxin-dependent glutamate synthase has been shown to contain one FMN but no FAD. The oxidation-reduction midpoint potentials of the FMN and the other prosthetic group, a [3Fe-4S]1+,0 cluster, have both been estimated to be -225 mV by cyclic voltammetry. Confirmation of the isopotential nature of the two prosthetic groups of the enzyme has been obtained using deazariboflavin phototitrations. Flash photolysis measurements have allowed determination of the second-order rate constants for reduction of both of the prosthetic groups of the enzyme by the 5-deazariboflavin semiquinone radical.

Amino Acid Oxidoreductases↗

PubMed1996

Axial heme ligation in the cytochrome bc1 complexes of mitochondrial and photosynthetic membranes. A near-infrared magnetic circular dichroism and electron paramagnetic resonance study.

The combination of EPR and low-temperature near-IR magnetic circular dichroism spectroscopies have been used to investigate the axial ligation of the cytochromes in the cytochrome bc1 complexes from bovine heart mitochondria, Rhodobacter capsulatus, Rhodobacter sphaeroides, and Rhodospirillum rubrum, and the purified cytochromes c1 from bovine heart mitochondria, Rb. capsulatus and Rb. sphaeroides. The possibility of axial ligation of cytochrome c1 by the amino terminus of the polypeptide was also assessed by acetylating the N-terminus of Rb. capsulatus cytochrome c1 and comparing the properties of the acetylated and unmodified samples. The results are consistent with bis-histidine axial ligation for the high- and low-potential b-type cytochromes and histidine/methionine axial ligation for the c1-type cytochrome in the intact cytochrome bc1 complexes. Purified samples of cytochrome c1 are mixtures of two forms, one with histidine/methionine and the other with bis-histidine axial ligation. The form with bis-histidine axial ligation is also assembled in the M183L mutant of the Rb. capsulatus cyt bc1 complex in which the methionine residue coordinating cyt c1 is replaced by a leucine. The bis-histidine form appears to be an artifact of dissociation of cytochrome c1 from the cytochrome bc1 complex and is greatly enhanced particularly in the bacterial cytochromes c1 by sample handling and the addition of 50% (v/v) ethylene glycol or glycerol.

Amino Acid Sequence↗

PubMed1995

The effect of N-bromosuccinimide on ferredoxin:NADP+ oxidoreductase.

Treatment of spinach leaf ferredoxin:NADP+ oxidoreductase (FNR) with N-bromosuccinimide (NBS), under conditions where approximately one tryptophan residue per enzyme was modified, resulted in a loss of between 80 and 85% of the activity of the enzyme when electron transfer from NADPH to either ferredoxin or 2,6-dichlorophenol-indophenol was measured. Amino acid analysis revealed no detectable modification by NBS of any FNR amino acids other than tryptophan. Complex formation with ferredoxin, but not with NADP+, prevented both the inhibition of activity and the modification of tryptophan caused by the treatment with NBS. Modification of one FNR tryptophan residue had no significant effect on the Km values of the enzyme for either ferredoxin or NADPH or on the binding constants for the FNR complexes with either ferredoxin or NADP+. NBS treatment had only very small effects on the absorbance and circular dichroism spectra of FNR and did not significantly affect either the oxidation-reduction midpoint potential of the FAD prosthetic group of the enzyme or inhibit the reduction of the FAD group by NADPH. These results raise the possibility that a tryptophan residue may play a role in the electron transfer between the FAD of FNR and the enzyme substrate, ferredoxin.

Bromosuccinimide↗

PubMed1995

The oxidation-reduction properties of spinach thioredoxins f and m and of ferredoxin:thioredoxin reductase.

Oxidation-reduction midpoint potentials have been determined, using cyclic voltammetry, for the active-site disulfide/dithiol couples of spinach thioredoxins f and m and of spinach ferredoxin:thioredoxin reductase (FTR) and for a component likely to be the [4Fe-4S] cluster of FTR. Values for the midpoint potentials (n = 2) of -210 +/- 10 mV were determined for both thioredoxins f and m. Two redox centers were detected in FTR, with midpoint potential values of -230 +/- 10 mV (n = 2) and +340 +/- 30 mV, respectively. Alkylation of the active-site cysteines of FTR by treatment of the enzyme with N-ethylmaleimide (NEM) eliminates the component with the -230 mV midpoint potential, allowing one to assign this value to the active site disulfide/dithiol couple. Inasmuch as the only other electron-carrying center known to be present in FTR is the [4Fe-4S] cluster, it appears likely that the high-potential component can be attributed to this redox moiety. The midpoint potential value of the high-potential feature shifts slightly, to +380 +/- 20 mV, in the NEM-treated enzyme.

Chloroplast Thioredoxins↗

PubMed1995

Ferredoxin-dependent CO2 fixation in bean sprouts.

Extracts of bean sprouts are capable of reducing ferredoxin and of catalyzing the incorporation of bicarbonate and acetyl coenzyme A into an organic compound that is likely to be pyruvate, in a reaction that requires reduced ferredoxin. The rate of the reaction, the first known for which ferredoxin appears to serve as the direct reductant for CO2 fixation in a higher plant, depends on the concentrations of both ferredoxin and bicarbonate, with half-maximal rates being observed at ferredoxin and bicarbonate concentrations of 0.8 microM and 200 microM, respectively.

Acetyl Coenzyme A↗

PubMed1994

Transient kinetic and oxidation-reduction studies of spinach ferredoxin:nitrite oxidoreductase.

The oxidation-reduction midpoint potentials for the two prosthetic groups of the chloroplast-located, ferredoxin-dependent nitrite reductase of spinach leaves have been determined by spectroelectrochemical titrations and cyclic voltammetry. The average of the results obtained by the two techniques are Em = -290 mV for the siroheme group and Em = -365 mV for the [4Fe-4S] cluster. The value obtained for the [4Fe-4S] cluster is substantially more positive than values obtained previously in experiments which utilized electron paramagnetic resonance spectroscopy at cryogenic temperatures to monitor the reduction state of the cluster. Laser flash photolysis experiments have been used to monitor electron transfer from reduced ferredoxin to nitrite reductase and have provided the first evidence for electron transfer between the two prosthetic groups of the enzyme. The effect of ionic strength on the observed kinetics has provided support for the proposal that electrostatic interactions between ferredoxin and nitrite reductase play an important role in the reaction mechanism.

Kinetics↗

PubMed1994

Spectroscopic characterization of flavocytochrome c-552 from the photosynthetic purple sulfur bacterium Chromatium vinosum.

The identities of the axial ligands to the two hemes of the flavocytochrome c-552 isolated from the photosynthetic purple sulfur bacterium Chromatium vinosum have been investigated by visible/near-infrared absorption and magnetic circular dichroism (MCD) spectroscopies, with parallel electron paramagnetic resonance (EPR) studies. One of the hemes has histidine and methionine as axial ligands and has a local environment that is relatively insensitive to the composition of the bulk medium. The second heme, the local environment of which is sensitive to changes in the composition of the bulk medium, exists as a mixture of two forms, only one of which has histidine/methionine axial ligation. On the basis of its EPR characteristics, the other form most likely has histidine/lysine axial ligation. In aqueous solution near neutral pH, more than half of the second heme is present as the histidine/lysine form, while in 50:50 water/ethylene glycol the histidine/methionine form is the dominant one.

Chromatium↗

PubMed1994

Cloning and sequencing of the gene encoding spinach ferredoxin-dependent glutamate synthase.

The nucleotide sequences of two cDNA clones, totalling 4948 bp in length, encoding 98% of the 1483 amino acids of the mature form of the ferredoxin-dependent glutamate synthase of spinach chloroplasts have been determined. The amino-terminal sequence of the enzyme has been determined by direct sequencing of the protein. The deduced amino-acid sequence of the spinach enzyme is 83% identical to that of the ferredoxin-dependent maize enzyme and shows significant sequence homology to two prokaryotic NAD(P)H-dependent glutamate synthases. Analysis of spinach genomic DNA indicates the presence of a single-copy gene for the spinach enzyme. Northern analysis reveals the presence of a single 5.5 kb transcript, which is present in higher levels in young spinach leaves than in older leaves.

Amino Acid Sequence↗

PubMed1994

Characterization of flavocytochrome C552 from the thermophilic photosynthetic bacterium Chromatium tepidum.

A M(r) 68 kDa flavocytochrome c552 has been isolated from the thermophilic photosynthetic purple sulfur bacterium Chromatium tepidum and shown to consist of a M(r) 25 kDa subunit that contains two covalently bound heme c and a M(r) 43 kDa subunit that probably contains a single FAD. The prosthetic group content, absorbance spectra, and subunit composition of the C. tepidum flavocytochrome are quite similar to those previously reported for the flavocytochrome c552 isolated from a mesophilic Chromatium species, Chromatium vinosum. The oxidation-reduction properties of the hemes present in the C. tepidum flavocytochrome have been characterized by titrations, the effect of temperature on the catalytic activity of the protein has been investigated, and the heme environment has been characterized using resonance Raman spectroscopy.

Chromatium↗

PubMed1993

Ferredoxin binding site on ferredoxin: NADP+ reductase. Differential chemical modification of free and ferredoxin-bound enzyme.

The chloroplast enzyme ferredoxin: NADP+ reductase (FNR) catalyzes the reduction of NADP+ by ferredoxin (Fd). FNR and Fd form a 1:1 complex that is stabilized by electrostatic interactions between acidic residues of Fd and basic residues of FNR. To localize lysine residues at the Fd binding site of FNR, the FNR:Fd complex (both proteins from spinach) was studied by differential chemical modification. In a first set of experiments, free FNR and the FNR:Fd complex were reacted with the N-hydroxysuccinimidyl ester of biotin. Biotinylated peptides and non-biotinylated peptides were separated on monovalent avidin-Sepharose and purified by high-performance liquid chromatography. Two peptides containing Lys18 and Lys153, respectively, were less biotinylated in complexed FNR than in free FNR. In a second set of experiments, free and complexed FNR were treated with 4-N,N-dimethylaminoazobenzene-4'-isothiocyano-2'-sulfonic acid (S-DABITC) to obtain coloured lysine-modified FNR. Protection of Lys153 was again found by modification with S-DABITC. In addition, Lys33 and Lys35 were less labelled in the S-DABITC-modified. Fd-bound enzyme. FNR modified in the presence, but not in the absence, of Fd was still able to bind Fd, indicating that the Fd-protected residues are involved in the formation of the Fd:FNR complex. The lysine residues disclosed by differential modification surround the positive end of the molecular dipole moment (558 Debye approximately 1.85 x 10(-27) Cm) and are located in a domain of strong positive potential on the surface of the FNR molecule. This domain we had proposed to belong to the binding site of FNR for Fd [De Pascalis, A. R., Jelesarov, I., Ackermann, F., Koppenol, W. H., Hirasawa, M., Knaff, D. B. & Bosshard, H. R. (1993) Protein Science 2. 1126-1135]. The prediction was based on the complementarity of shape between positive and negative potential domains of FNR and Fd, respectively.

Amino Acid Sequence↗

PubMed1993

The interaction between cytochrome c2 and the cytochrome bc1 complex in the photosynthetic purple bacteria Rhodobacter capsulatus and Rhodopseudomonas viridis.

The rates of electron transfer from a ubiquinol analogue to cytochrome c2 catalyzed by the cytochrome bc1 complexes of Rhodobacter capsulatus and Rhodopseudomonas viridis were measured as a function of ionic strength. The effects of ionic strength on the kinetic parameters for the reactions are consistent with a role for electrostatic complex formation between cytochrome c2 and the cytochrome bc1 complex in the electron-transfer pathways in both photosynthetic purple non-sulfur bacteria. Additional support for a docking model in which positively charged lysines on cytochrome c2 interact with negatively charged groups on the Rb. capsulatus cytochrome bc1 complex was obtained from kinetic experiments using Rb. capsulatus cytochrome c2 and equine cytochrome c in which specific lysine residues were altered by site-directed mutagenesis and chemical modification, respectively. Equine cytochrome c, which is a poor electron donor to the reaction center of Rps. viridis, is an effective electron acceptor for the Rps. viridis cytochrome bc1 complex. Chemical modification of lysine residues on Rps. viridis cytochrome c2 has a substantially greater effect on the reduction of the Rps. viridis reaction center by ferrocytochrome c2 than on the oxidation of the Rps. viridis cytochrome bc1 complex by ferricytochrome c2. These data suggest that the docking site for Rps. viridis cytochrome c2 on the Rps. viridis reaction center tetraheme subunit differs in structure from the docking site for the cytochrome on the Rps. viridis cytochrome bc1 complex to a significant extent. In this respect, Rps. viridis differs from photosynthetic purple non-sulfur bacteria in which the reaction center does not contain a tetraheme subunit, where the binding sites for cytochrome c2 on the reaction center and the cytochrome bc1 complex appear to be quite similar.

Chromatography, Gel↗

PubMed1993

Characterization of two low-potential cytochromes from bean sprouts.

Two cytochromes have been isolated from chlorophyll-free bean sprouts, purified and characterized. The more abundant cytochrome was purified to apparent homogeneity and exhibits visible region absorbance maxima at 416, 520 and 550 nm in the reduced form and at 410 and 530 nm in the oxidized form. Although Resonance Raman spectra of this cytochrome closely resemble those of c-type cytochromes, pyridine hemochromogen analysis suggests that this cytochrome may contain a variant of heme c as its prosthetic group. The cytochrome has an apparent molecular mass of 12.5 kDa, an isoelectric point > 9.0 and a midpoint oxidation-reduction potential (Em) of -130 mV at pH 8.0. The less abundant of the two cytochromes, which was not completely purified, exhibits absorbance maxima at 438 and 560 nm in the reduced form and at 411 nm in the oxidized form and was shown to contain heme c as a prosthetic group. This cytochrome, which may also contain FAD, has an apparent molecular mass of approx. 38 kDa, an isoelectric point > 9.0 and Em = -300 mV. Preliminary results indicate that both cytochromes can form electrostatically-stabilized complexes with ferredoxin, suggesting the possibility that one or both of the cytochromes may participate in low-potential, non-photosynthetic electron transfer pathways involving ferredoxin.

Circular Dichroism↗

PubMed1993

Binding of ferredoxin to ferredoxin:NADP+ oxidoreductase: the role of carboxyl groups, electrostatic surface potential, and molecular dipole moment.

The small, soluble, (2Fe-2S)-containing protein ferredoxin (Fd) mediates electron transfer from the chloroplast photosystem I to ferredoxin: NADP+ oxidoreductase (FNR), a flavoenzyme located on the stromal side of the thylakoid membrane. Ferredoxin and FNR form a 1:1 complex, which is stabilized by electrostatic interactions between acidic residues of Fd and basic residues of FNR. We have used differential chemical modification of Fd to locate aspartic and glutamic acid residues at the intermolecular interface of the Fd:FNR complex (both proteins from spinach). Carboxyl groups of free and FNR-bound Fd were amidated with carbodiimide/2-aminoethane sulfonic acid (taurine). The differential reactivity of carboxyl groups was assessed by double isotope labeling. Residues protected in the Fd:FNR complex were D-26, E-29, E-30, D-34, D-65, and D-66. The protected residues belong to two domains of negative electrostatic surface potential on either side of the iron-sulfur cluster. The negative end of the molecular dipole moment vector of Fd (377 Debye) is close to the iron-sulfur cluster, in the center of the area demarcated by the protected carboxyl groups. The molecular dipole moment and the asymmetric surface potential may help to orient Fd in the reaction with FNR. In support, we find complementary domains of positive electrostatic potential on either side of the FAD redox center of FNR. The results allow a binding model for the Fd:FNR complex to be constructed.

Amino Acid Sequence↗