Henry Kamin

[92] The preparation and properties of microsomal TPNH-cytochrome c reductase from pig liver

Publisher Summary This chapter discusses the preparation and properties of microsomal TPNH-cytochrome c reductase from pig liver. It is purified from microsomes both as a partially purified microsomal subparticle, and with lipase treatment and fractionation, as a soluble flavoprotein essentially homogeneous in the ultracentrifuge. The assay depends upon measurement of the rate of cytochrome c reduction at 550 mμ. Cytochrome c reductase activity in microsomes is associated with marked TPNH-neotetrazolium diaphorase activity. This activity is disproportionately lost upon lipase treatmeat of microsomes, and its loss may serve as an indication of loss of an intermediate cofactor in the intact microsome, or of loss of a specific environmental or configurational state of the enzyme. The steps of purification procedure described are: preparation of lipase, preparation of microsomes, lipase solubilization, pH precipitation, ammonium sulfate fractionation, column chromatography on hydroxylapatite, and calcium phosphate gel concentration. The prosthetic group of TPNH-cytochrome c reductase enzyme is flavin adenine dinucleotide, but the apoenzyme can be reactivated by FMN and FAD. The enzyme is specific for TPNH, but is relatively nonspecific for electron acceptor. TPNH-cytochrome c reductase has a pH optimum of 8.2, which appears to be independent of the buffer used. TPNH-cytochrome c reductase is markedly stimulated by low levels of p-chloromercuribenzoate; maximal activation is reached at 2 moles of PCMB per mole of flavin, and higher concentrations inhibit.

Steroidogenic electron transport in adrenal cortex mitochondria

SummaryThe flavoprotein NADPH-adrenodoxin reductase and the iron sulfur protein adrenodoxin function as a short electron transport chain which donates electrons one-at-a-time to adrenal cortex mitochondrial cytochromes P-450. The soluble adrenodoxin acts as a mobile one-electron shuttle, forming a complex first with NADPH-reduced adrenodoxin reductase from which it accepts an electron, then dissociating, and finally reassociating with and donating an electron to the membrane-bound cytochrome P-450 (Fig. 9). Dissociation and reassociation with flavoprotein then allows a second cycle of electron transfers. A complex set of factors govern the sequential protein-protein interactions which comprise this adrenodoxin shuttle mechanism; among these factors, reduction of the iron sulfur center by the flavin weakens the adrenodoxinadrenodoxin reductase interaction, thus promoting dissociation of this complex to yield free reduced adrenodoxin. Substrate (cholesterol) binding to cytochrome P-450scc both promotes the binding of the free adrenodoxin to the cytochrome, and alters the oxidation-reduction potential of the heme so as to favor reduction by adrenodoxin. The cholesterol binding site on cytochrome P-450scc appears to be in direct communication with the hydrophobic phospholipid milieu in which this substrate is dissolved. Specific effects of both phospholipid headgroups and fatty acyl side-chains regulate the interaction of cholesterol with its binding side. Cardiolipin is an extremely potent positive effector for cholesterol binding, and evidence supports the existence of a specific effector lipid binding site on cytochrome P.450scc to which this phospho-lipid binds.

Uncoupling of oxygen activation from hydroxylation in a bacterial salicylate hydroxylase

Benzoate stimulates DPNH oxidation in a bacterial flavoprotein, salicylate hydroxylase (salicylate, DPNH : oxygen oxidoreductase (1 - hydroxylating, 1-decarboxylating)) with a higher Km than that of salicylate. Whereas salicylate is hydroxylated to catechol, with formation of H2O, benzoate is unchanged and hydrogen peroxide is released during the course of DPNH oxidation. Vmax for salicylate hydroxylation and benzoate-stimulated DPNH oxidation are identical. The results suggest that benzoate binds to the salicylate site as a pseudosubstrate and “uncouples” oxygen activation from hydroxylation.

An iron tetrahydroporphyrin prosthetic group common to both assimilatory and dissimilatory sulfite reductases

The heme† chromophore of the “assimilatory” E. coli sulfite reductase is an iron-octacarboxylic tetrahydroporphyrin of the isobacteriochlorin type (1). Although the two “dissimilatory” sulfite reductases, desulfoviridin and desulforubidin, from the sulfate reducing bacteria Desulfovibrio gigas and Desulfovibrio desulfuricans (Norway strain), have absorption spectra and reaction products which differ from those of E. coli sulfite reductase, the present studies indicate that they contain prosthetic groups with an organic structure closely similar or identical to that of the E. coli sulfite reductase heme. EPR spectra show high-spin ferriheme in all three enzymes. It is clear, however, that the prosthetic groups must reside in substantially different environments within their respective proteins.

[46] Siroheme: Methods of Isolation and characterization

Publisher Summary This chapter describes the methods of characterization and isolation related to siroheme. A number of sulfite and nitrite reductase enzymes from bacteria, fungi, and plants are found to contain a new type of heme-related prosthetic group that is known as siroheme. Certain of these enzymes are associated in vivo with phosphorylating respiratory chains and are therefore presumably normally membrane bound. Most of the enzymes studied, however, serve a purely biosynthesic role in the assimilation of sulfate and nitrate; there is no evidence for membrane association of these enzymes. Although siroheme enzymes generally yield siroheme itself upon extraction with acetone/HCl, the desulfoviridin-type of sulfite reductase from Desulfovibrio gigas and D. vulgaris yields sirohydrochlorin under such conditions. The properties of sirohydrochlorin compared with those of siroheme in media used during the extraction and purification of the latter compound from enzyme systems are described by Murphy and Siegel.

REDUCTION OF MICROSOMAL CYTOCHROMES BY PYRIDINE NUCLEOTIDES.

Summary 1. Reduction of microsomal cytochromes by TPNH and DPNH in whole microsomes and in a solubilized fraction has been studied. 2. Difference spectra, and kinetic analysis of the cycle of reduction of cytochrome b 5 by DPNH and TPNH and reoxidation by air, show that reduction of this cytochrome by DPNH is more rapid than by TPNH. The rate of reoxidation of reduced cytochrome b 5 is relatively independent of which nucleotide serves as reductant. Aerobic disappearance of DPNH is more rapid than that of TPNH; TPNH oxidation is inhibited by TPN + . 3. A “solubilized preparation” from lipase (EC 3.1.1.3)-treated microsomes 10 catalyzes reduction of its endogenous cytochrome b 5 by TPNH or DPNH, but TPNH-cytochrome b6 reductase activity has been disproportionately lost as compared to TPNH-cytochrome c reductase (EC 1.6.2.3) activity. TPNH cannot reduce externally-added purified cytochrome b 5 , while DPNH can reduce it partially. 4. Carbon monoxide partially inhibits cytochrome bs reduction by TPNH in the “solubilized fraction”; little effect on DPNH reduction of this cytochrome was observed. 5. Menadione stimulates TPNH disappearance catalyzed by both “solubilized preparation” and purified TPNH-cytochrome c reductase. In addition, reoxidation of reduced cytochrome b 5 is stimulated by this quinone; this oxidation may be due to peroxide formation. 6. Possible relationships between TPNH-cytochrome c reductase, cytochrome b 5 , the carbon-monoxide binding cytochrome, and microsomal hydroxylation reactions are discussed in the light of present findings and those of other laboratories.

[52] Salicylate hydroxylase

Publisher Summary This chapter provides information on enzyme salicylate hydroxylase. Salicylate hydroxylase is a flavoprotein hydroxylase, a member of a class of enzymes elaborated by some soil bacteria, typically pseudomonads. These enzymes are induced by the presence of phenolic compounds. The activity of the enzyme was determined spectrophotometrically by following the rate of substrate-dependent oxidation of NADH in a Cary 14 or Gilford 2400 spectrophotometer at 340 nm. Salicylate hydroxylate is established as a flavoprotein containing flavin adenine dinucleotide (FAD) as the sole prosthetic group. The purified hydroxylase migrates as one homogeneous and symmetrical peak in the Model E analytical untracentrifuge with 0.1 M KCl as solvent. Enzyme activity can also be assayed by measuring the rate of oxygen consumption using an oxygen electrode. This enzyme, in addition to its FAD prosthetic group, has three substrates: salicylate or other aromatic compound, pyridine nucleotide, and oxygen. Its kinetics, therefore, can be expected to be complex.

Flavin interaction in NADPH-sulfite reductase.

E. coli NADPH-sulfite reductase, depleted of FMN but retaining its FAD, has been prepared by photoirradiation of native enzyme in 30% — saturated ammonium sulfate. FMN-depleted enzyme loses its ability to reduce (using NADPH) ferricyanide, cytochrome c, sulfite, or the enzyme’s own heme-like chromophore. However, the FAD remains rapidly reducible by NADPH, and the FMN-depleted enzyme retains NADPH-acetylpyridine NADP* transhydrogenase activity. Thus, FAD can serve as entry port for NADPH electrons, and FMN is required for further transmission along the enzyme’s electron transport chain. These data, plus other studies, have enabled us to suggest a mechanism for catalysis which involves FAD cycling between the fully-oxidized and fully-reduced forms while FMN cycles between fully-reduced and semiquinone. This mechanism, which includes a disproportionation step, permits a “step-down” from the twoelectron donor, NADPH, to a succession of equipotential one-electron transfer steps.

[209] TPNH-sulfite reductase (Escherichia coli)☆☆☆★

Publisher Summary This chapter discusses the methods of preparation of TPNH-Sulfite Reductase ( Escherichia coli ). Sulfite reductase is a key enzyme on the regulated pathway of cysteine biosynthesis in the enterobacteria, and the level of enzymatic activity is closely responsive to the intracellular cysteine content. It is, therefore, possible to derepress enterobacteria ior sulfite reductase by growth on limiting sulfur sources, so that under favorable conditions the enzyme can comprise approximately 0.5% of the soluble cell protein. Sulfite reductases from enterobacteria and yeast are high molecular weight iron-flavoproteins that can utilize either TPNH or artificial dyes (such as reduced methyl viologen) as donors for the six-electron reduction of sulfite to sulfide. A unit of activity is defined as that amount of enzyme that catalyzes the oxidation of 1 micromole of TPNH per minute with sulfite as electron acceptor under the above assay conditions. The purified enzyme appears homogeneous by sedimentation analysis in the Beckman Model E ultracentrifuge and by electrophoresis in both polyacrylamide gel (pH 9) and cellulose acetate strip (pH 7.7) supporting media.

Indoleacetic acid and growth of bacteria with varying requirements for nicotinic acid and tryptophane.

Summary and Conclusions 1. A number of organisms whose tryptophane and nicotinic acid requirements are known were tested for possible inhibition by indoleacetic acid and reversal by nicotinic acid and tryptophane in chemically defined media. 2. Escherichia coli, Lactobacillus arabinosus 17-5, L. casei, Streptococcus faecalis R, and Aerobacter aerogenes were inhibited by concentrations of indoleacetic acid of one mg/ml Occasionally slight inhibitions were noted at 0.1 mg/ml levels. 3. No significant relief of inhibition was obtained with either nicotinic acid or tryptophane 4. Staphylococcus aureus and Mycobacterium tuberculosis (607) were not inhibited by indoleacetic acid at the concentrations studied. 5. Fairll consistent stimulation at 0.1 mg/ml levels of indoleacetic acid were obtained with L. arabinosus and L. casei in the presence of limiting concentrations of tryptophane. 6. The significance of these results in relocation to possible metabolite-antagonist relationship between indoleacetic acid and tryptophane or nicotinic acid is discussed.