Spectroscopic studies of protein-heme interactions accompanying the allosteric transition in methemoglobins

Resonance Raman, optical absorption, and circular dichroism spectroscopic techniques have been used to examine the effect of the addition of inositol hexaphosphate (IHP) to a series of carp and human methemoglobin derivatives. Markers of spin equilibrium in the high-frequency region (1450-1650 cm-1) of the resonance Raman spectrum yield high/low-spin ratios consistent with direct magnetic susceptibility measurements. Changes in the low-frequency region (100-600 cm-1) of the resonance Raman spectrum appear to correlate with the quaternary structure transition. Changes in the ultraviolet absorption spectra and the circular dichroism spectra also appear to be related to the quaternary structure change. By using the resonance Raman spin markers, we find that those derivatives of carp methemoglobin which are in spin equilibrium have a larger ratio of high-spin to low-spin populations than the corresponding derivatives of human methemoglobin. Upon the addition of IHP to the methemoglobins the spin equilibrium is shifted toward a larger high-spin population. This change in equilibrium is larger for the carp protein than for the human protein. We obtain an IHP-induced change in the free energy difference between the high-spin and low-spin states of 300 cal/mol for those human methemoglobins in which a quaternary structure change occurs and 600 cal/mol for carp methemoglobins. Our data are consistent with a quaternary structure change induced by IHP in all the carp methemoglobins studied (F-, H2O, SCN-, NO2-, N3-, and CN-) and in the F-, H2O, and SCN- derivatives of the human protein but not in the NO2-, N3-, and CN- derivatives. The Fe-CN stretching mode has been identified by isotopic substitution and found to be unchanged in frequency in carp CN- metHb when the quaternary structure is changed. On the basis of our results we conclude that the protein forces at the heme due to the addition of IHP do not significantly affect the position of the iron atom with respect to the heme plane. Rather, the changes in spin equilibrium may be caused by protein-induced changes in the orientation of the proximal histidine or tertiary structure changes in the heme pocket which affect the porphyrin macrocycle. Either of these changes, or a combination thereof, leads to changes in the iron d orbital energies and concomitant changes in the spin equilibrium.     

Single-channel analysis of a potassium inward rectifier in myocytes of newborn rat heart

Summary Unitary K⁺ currents in single cells isolated from ventricular muscle of newborn rat hearts were measured in response to different potentials and [K] _o . TheI/V curves were linear for potentials more negative than the zero-current voltage: especially in high [K] _o (150nm KCl), no clear outward currents could be detected indicating a drastic rectification in the inward direction. The channel is mainly selective to K⁺ but Na⁺ ions are also carried (P _Na/P _K=0.056). The channel conductance is proportional to the square root of [K] _o but Na⁺ ions seem to have a facilitatory effect on _K, the single-channel conductance. The channel activity, measured asP _o, i.e. the probability to find the channel in open state, decreased as the membrane was hyperpolarized. This behavior was tentatively explained by an inactivation process as the membrane becomes more negative. The rate constants of the transitions between the different states were calculated according to a C–O–C model. A control of the gating process by permeant ion K⁺ was postulated, based on the increase of one of the rate constants from the closed to the open state with [K] _o . Finally, the macroscopicI/V curves calculated fromP _o and i, the unit current, were found to be characteristic of a ion-blocked inward rectifier.     

Effects of dicyclohexane derivatives on androgen metabolism in the rat prostate

Dicyclohexane derivatives, which inhibit the binding of testosterone and dihydrotestosterone (DHT) to the androgen-binding protein (ABP) of rat epididymis without interfering with their binding to the androgen receptor, show a similar selectivity in their effects on androgen metabolism. Their ability to inhibit the aromatization of testosterone has been reported previously. This paper demonstrates that they are potent inhibitors of 3 alpha(beta)-hydroxysteroid:NAD(P)+ oxidoreductase activity (3-HSD) in the particulate fraction from rat prostate gland; the values of Ki for their inhibition of this enzyme are similar to that of the Km for DHT as substrate. The dicyclohexane derivatives are markedly less effective against the cytosolic NADPH-dependent 3-HSD, and they do not appear to inhibit testosterone 5 alpha-reductase activity. These characteristics are likely to complicate the proposed use of the dicyclohexane derivatives as probes for the role of ABP in vivo. However, they may be of interest in the study of structure-activity relationships in androgen-metabolizing enzymes, particularly in the examination of the different forms of 3-HSD.     

Radioimmunoassay of ochratoxin A in barley

A simple and rapid radioimmunoassay was developed for the quantitative determination of ochratoxin A in barley. [14C]ochratoxin A, with a specific activity of 130 Ci/mol, was used as the tracer. Toxin levels below 100 ng/ml required a cleanup step. Three methods (the Association of Official Analytical Chemists cleanup method, the solvent partition method, and the Extrelut 3 column cleanup method) were compared.     

Resonance Raman spectra of the heme in leghemoglobin. Evidence for the absence of ruffling and the influence of the vinyl groups

Resonance Raman spectra of deoxy and carbonmonoxy leghemoglobin (Lb) are compared to the corresponding forms of human adult hemoglobin (HbA). It is found that the heme "core size" indicator line has nearly the same frequency for the two deoxyhemoglobins and the pi-electron density-sensitive line also falls at the same frequency. However, several other modes occur at very different frequencies in the spectra of the two proteins. From an examination of the spectrum of an HbA derivative in which the beta-carbon atoms of the heme vinyl groups were deuterated, it appears that the major differences between deoxy-HbA and -Lb may result from conformational changes in the vinyl groups. No evidence for the suggested ruffling (Irwin, M. J., Armstrong, R. S., and Wright, P. E. (1981) FEBS Lett. 133, 239-243) in deoxy-Lb was found. The spectra of carbonmonoxy-Lb and -HbA were also found to be very different. As in the deoxy case, some of these frequency differences could be attributed to vinyl group conformational differences. However, from the large difference in the pi-electron density-sensitive line, it appears that the vinyl pi-conjugation into the porphyrin in Lb(CO) may be different than it is in HbA(CO). The vinyl conformational differences may be a consequence of the looser heme pocket in Lb than in HbA. The difference in pi-conjugation could make a significant contribution to the difference in ligand binding affinity for these two globins.     

Structural characterization of cytochrome c peroxidase by resonance Raman scattering

Resonance Raman scattering studies are reported on freshly prepared and aged ferric, ligand-free ferrous, and CO-bound ferrous cytochrome c peroxidase. The ferric form of the fresh enzyme has a heme which is penta-coordinate high spin, independent of buffer over the pH range 4.3-7, as determined by well established Raman marker lines. The aged enzyme displays a mixture of spin and coordination states, but it can be stabilized in the penta-coordinate high spin form in the presence of phosphate. These results can be accounted for by considering the size of the channel (6 A wide, 11 A long) between the distal side of the heme and the outer surface of the protein. A phosphate ion may be accommodated in this channel resulting in the stabilization of the distal heme pocket. The ferrous cytochrome c peroxidase in both the ligand-free and CO-bound states has an acidic and an alkaline form. The acidic form has the characteristic spectral features of peroxidases: a high frequency iron-histidine stretching mode (248 cm-1), a high frequency Fe-CO stretching mode (537 cm-1), and a low frequency C-O stretching mode (1922 cm-1). At alkaline pH these frequencies become similar to those of hemoglobin and myoglobin, with the corresponding modes located at 227, 510, and 1948 cm-1, respectively. We attribute the acid/alkaline transition in the ferrous forms of cytochrome c peroxidase to a rearrangement mainly of the proximal side of the heme, culminating in a change of steric interactions between the proximal histidine and the heme or of the hydrogen bonding network involving the proximal histidine. The new data presented here reconcile many inconsistencies reported in the past.     

Review: Biocatalytic transformations of ferulic acid: An abundant aromatic natural product

In this review we examine the fascinating array of microbial and enzymatic transformations of ferulic acid. Ferulic acid is an extremely abundant, preformed phenolic aromatic chemical found widely in nature. Ferulic acid is viewed as a commodity scale, renewable chemical feedstock for biocatalytic conversion to other useful aromatic chemicals. Most attention is focused on bioconversions of ferulic acid itself. Topics covered include cinnamoyl side-chain cleavage; nonoxidative decarboxylation; mechanistic details of styrene formation; purification and characterization of ferulic acid decarboxylase; conversion of ferulic acid to vanillin;O-demethylation; and reduction reactions. Biotransformations of vinylgualacol are discussed, and selected biotransformations of vanillic acid including oxidative and nonoxidative decarboxylation are surveyed. Finally, enzymatic oxidative dimerization and polymerization reactions are reviewed.     

A comparative study of glucose conversion to ethanol by Zymomonas mobilis and a recombinant Escherichia coli

Summary Zymomonas mobilis and recombinant Escherichia coli B (pLOI297) were compared in side-by-side batch fermentations using a synthetic cellulose hydrolysate (glucose/salts) medium with pH control at 6.0 and an inoculation cell density of 35–50 mg dry wt. cells/L. At a nominal glucose concentration of 6%, both cultures achieved near maximal theoretical ethanol yields; however, the Z. mobilis fermentation was complete at 13h compared to 33h for the E.coli fermentation. With approx.12% glucose, the Z. mobilis fermentation was complete in 20h with a process yield of 0.49 g ethanol/g added glucose compared to the E. coli fermentation which remained 20% incomplete after 6 days resulting in a process yield of only 0.32 g/g. Nutrient supplementation (10g tryptone/L) resulted in complete fermentation of 12% glucose (pH 6.3) by the recombinant E. coli in 4 days, with a yield of 0.48 g/g.     

Evidence for the involvement of PSI-E subunit in the reduction of ferredoxin by photosystem I.

Of the stroma-accessible proteins of photosystem I (PSI) from Synechocystis sp. PCC 6803, the PSI-C, PSI-D and PSI-E subunits have already been characterized, and the corresponding genes isolated. PCR amplification and cassette mutagenesis were used in this work to delete the psaE gene. PSI particles were isolated from this mutant, which lacks subunit PSI-E, and the direct photoreduction of ferredoxin was investigated by flash absorption spectroscopy. The second order rate constant for reduction of ferredoxin by wild type PSI was estimated to be approximately 10(9) M-1s-1. Relative to the wild type, PSI lacking PSI-E exhibited a rate of ferredoxin reduction decreased by a factor of at least 25. After reassociation of the purified PSI-E polypeptide, the original rate of electron transfer was recovered. When a similar reconstitution was performed with a PSI-E polypeptide from spinach, an intermediate rate of reduction was observed. Membrane labeling of the native PSI with fluorescein isothiocyanate allowed the isolation of a fluorescent PSI-E subunit. Peptide analysis showed that some residues following the N-terminal sequence were labeled and thus probably accessible to the stroma, whereas both N- and C-terminal ends were probably buried in the photosystem I complex. Site-directed mutagenesis based on these observations confirmed that important changes in either of the two terminal sequences of the polypeptide impaired its correct integration in PSI, leading to phenotypes identical to the deleted mutant. Less drastic modifications in the predicted stroma exposed sequences did not impair PSI-E integration, and the ferredoxin photoreduction was not significantly affected. All these results lead us to propose a structural role for PSI-E in the correct organization of the site involved in ferredoxin photoreduction.