M?ssbauer investigations of high-spin ferrous heme proteins. I. Cytochrome P-450.

Anaerobically reduced samples of cytochrome P-450 from Pseudomonas putida were studied by M?ssbauer spectroscopy. In the presence of an applied magnetic field the high-spin ferrous heme iron showed an intricate pattern of electric and magnetic hyperfine interactions which could be parametrized successfully in terms of a spin Hamiltonian formalism. The results imply a very low (triclinic) symmetry of the heme iron. The effects of the ligand environment and of spin-orbit coupling result in a large zero-field splitting of the electronic ground state. The electronic ground state. The electric-field gradient tensor is characterized by a large asymmetry parameter, and its principal axes are rotated substantially from the frame that defines the zero-field splitting. This study shows that high-field M?ssbauer spectroscopy provides a unique tool for structural investigations of high-spin ferrous compounds and can substitute, under suitable conditions, for magnetic susceptibility measurements. The present paper focuses on the methodology and data analysis; in the subsequent paper the data obtained for P-450 are compared with new results obtained for hemoglobin, chloroperoxidase, and horseradish peroxidase.     

I. Mähren

Ohne ZusammenfassungDas Referat umfasst den Zeitraum vom 1. October 1890 bis 1. April 1891.     

The Anatomy of the Tarsi of Schistocerca gregaria Forskål

Summary The tarsus of S. gregaria is divided into three units (here called segments) and an arolium set between a pair of claws. The first segment bears three pairs of pulvilli in the fore and middle legs, and one pair and two single pulvilli in the hind legs. Segment two bears a pair of pulvilli, segment three one long pulvillus and the arolium a similar pad on the undersurface. The outer layers of the arolium pad differ from those of the pulvilli in possibly lacking an epicuticle and in having a layer of cuticle which, unlike the corresponding layer in the pulvilli, does not stain with protein stains. The claws and dorsal surfaces bear trichoid sensilla, basiconic sensilla and campaniform sensilla. Smaller basiconic sensilla and canal sensilla occur on the proximal part of the pulvilli, and basiconic sensilla on the arolium undersurface. Internally the cuticle is modified in the arolium and pulvilli so that rods of probably chitin and resilin are formed. This would impart flexibility to the undersurfaces whilst retaining some degree of rigidity which might prevent damage to the small and delicate sense organs on the pulvilli. The tip of the arolium is specialised for adhesion, and there are two large neurones internally which could conceivably monitor attachment or detachment of the tip. There are chordotonal organs in segment three, and several other large neurones throughout the tarsus, some of which are associated with the slings of tissue holding the apodeme in a ventral position. Gland cells occurring in the dorsal epidermis of the adult mature male are also briefly described.     

The CO adduct of yeast cytochrome c oxidase. M?ssbauer and photolysis studies

M?ssbauer spectra of 57Fe-enriched NADH-reduced yeast cytochrome c oxidase reveal two quadrupole doublets of unequal intensity; one (approximately 33%) is typical of high-spin ferrous heme with histidine coordination and is assigned to heme a3, while the other (approximately 67%) is typical of low-spin heme with two nitrogeneous axial ligands as expected from heme a. The excess intensity (approximately 17%) of the low-spin doublet must therefore be assigned to heme a3 in a modified environment. The M?ssbauer spectra of the same sample exposed to CO show that 50% of the heme iron forms a CO adduct, consistent with heme a3 being inhibited by CO. While low-spin hem a has the same M?ssbauer parameters as in the reduced sample, its intensity has dropped to 35%. A distinctly new high-spin species (approximately 15%) is observed and assigned to heme a in a modified environment. The comparable size of the unexpected high-spin heme a fraction in the CO adduct and the low-spin heme a3 fraction in the reduced enzyme suggest that they arise from the same material. This material is likely to be the inactive fraction that has been found in all preparations of resting yeast cytochrome c oxidase (Siedow, J.N., Miller, S., and Palmer, G. (1981) J. Bioenerg. Biomembr. 14, 171-179). The kinetics of CO recombination following photolysis of the CO complex further confirms the coexistence of two distinct fractions associated with active and inactive protein. The majority (approximately 74%), presumably active protein, recombines exponentially from 160 to 270 K following an Arrhenius law. The large activation enthalpy, delta H approximately 35 kJ/mol, is comparable to that found in the beef heart enzyme, suggesting that the flashed-off CO is bound by the nearby CuB as in the mammalian system (Fiamingo, F.G., Altschuld, R.A., Moh, P.P., and Alben, J.O. (1982) J. Biol. Chem. 250, 1639-1650). In the minority, presumably inactive, fraction the CO recombination has fast nonexponential kinetics with a distribution of activation enthalpies peaking near delta Hp = 13 kJ/mol reminiscent of CO binding to myoglobin. In this inactive fraction CuB is apparently not accessible to the flashed-off CO.     

The role of Cas8?in type?I CRISPR interference

CRISPR (clustered regularly interspaced short palindromic repeat) systems provide bacteria and archaea with adaptive immunity to repel invasive genetic elements. Type I systems use ‘cascade’ [CRISPR-associated (Cas) complex for antiviral defence] ribonucleoprotein complexes to target invader DNA, by base pairing CRISPR RNA (crRNA) to protospacers. Cascade identifies PAMs (protospacer adjacent motifs) on invader DNA, triggering R-loop formation and subsequent DNA degradation by Cas3. Cas8 is a candidate PAM recognition factor in some cascades. We analysed Cas8 homologues from type IB CRISPR systems in archaea Haloferax volcanii (Hvo) and Methanothermobacter thermautotrophicus (Mth). Cas8 was essential for CRISPR interference in Hvo and purified Mth Cas8 protein responded to PAM sequence when binding to nucleic acids. Cas8 interacted physically with Cas5–Cas7–crRNA complex, stimulating binding to PAM containing substrates. Mutation of conserved Cas8 amino acid residues abolished interference in vivo and altered catalytic activity of Cas8 protein in vitro. This is experimental evidence that Cas8 is important for targeting Cascade to invader DNA.     

Ecole d'été de l'A.C.E.M.I.—Ha?ti

Six Canadian medical students record their experience at a summer school of tropical medicine in Haiti, sponsored by the Canadian Association of Medical Students and Internes. The social, economic and medical background is described, including “Voodoo” practices, language and Haitian art. Attention is directed to the occurrence of umbilical tetanus, diarrhea and malnutrition. From even a brief stay in a country such as Haiti one comes to appreciate that a public health program in an underdeveloped nation is not strictly a medical undertaking but must be seen in its social and economic contexts.     

M?ssbauer studies of adrenodoxin. The mechanism of electron transfer in a hydroxylase iron–sulphur protein

1. Mössbauer spectra were measured of adrenodoxin purified from porcine adrenal glands. They show similarities to the spectra of the plant ferredoxins. All of these proteins contain two atoms of iron and two of inorganic sulphide per molecule, and on reduction accept one electron. 2. As with the plant ferredoxins the adrenodoxin for these measurements was enriched with ⁵⁷Fe by reconstitution of the apo-protein, and subsequently was carefully purified and checked by a number of methods to ensure that it was in the same conformation as the native protein and contained no extraneous iron. 3. The Mössbauer spectra of oxidized adrenodoxin at temperatures from 4.2°K to 197°K show that the iron atoms are probably high-spin Fe³⁺, and in similar environments, and experience little or no magnetic field from the electrons. 4. Mössbauer spectra of reduced adrenodoxin showed magnetic hyperfine structure at all temperatures from 1.7°K to 244°K, in contrast with the reduced plant ferredoxins, which showed it only at lower temperatures. This is a consequence of a longer electron-spin relaxation time in reduced adrenodoxin. 5. At 4.2°K in a small magnetic field the spectrum of reduced adrenodoxin shows a sixline Zeeman pattern due to Fe³⁺ superimposed upon a combined magnetic and quadrupole spectrum due to Fe²⁺. 6. In a large magnetic field (30kG) each hyperfine pattern is further split into two. Analysis of these spectra at 4.2°K and 1.7°K shows that the effective fields at the Fe³⁺ and Fe²⁺ nuclei are in opposite directions. This agrees with the proposal, first made for the ferredoxins, that the iron atoms are antiferromagnetically coupled. 7. In accord with the model for the ferredoxins, it is proposed that the oxidized adrenodoxin contains two high-spin Fe³⁺ atoms which are antiferromagnetically coupled; on reduction one iron atom becomes high-spin Fe²⁺.     

The α-glucosidases of Dictyostelium discoideum: I. Identification and characterization

We have identified four isozymes of α-glucosidase in the cellular slime mold, Dictyostelium discoideum. The isozymes can be distinguished by their physical and enzymatic properties. α-Glucosidase-1, α-glucosidase-2, and α-gluocosidase-3 are all present in vegetative cells, while α-glucosidase-4 is present only after the cells have proceeded through aggregation. Three of the four enzymes, α-glucosidase-1, α-glucosidase-3, and α-glucosidase-4, have acidic pH optima of 3.5, 2.2, and 4.0, respectively. In contrast, α-glucosidase-2 has a neutral pH optimum, 7.25. α-Glucosidase-1, α-glucosidase-2, and α-glucosidase-3 are distinguishable by electrophoresis in native polyacrylamide gels. α-Glucosidase-4 comigrates with α-glucosidase-2 on native gels but they can be resolved by isoelectric focusing. The isozymes also differ with respect to affinity for the substrates p-nitrophenyl-α-d-glucoside and 4-methyl-umbelliferyl-α-d-glucopyranoside and the relative maximal rates of hydrolysis of these substrates. α-Glucosidases-1, -2, and -4 have apparent K_m's in the millimolar range while the apparent K_m of α-glucosidase-3 for p-nitrophenyl-α-d-glucoside is much higher. This may suggest that isozyme 3 is an endoglycosidase or may have greater affinity for other sugar substrates. α-Glucosidase-1 is the major isozyme in vegetative cells.     

The importance of quantitative M?ssbauer spectroscopy of MoFe-protein from Azotobacter vinelandii

The M?ssbauer spectra of MoFe-protein of Azotobacter vinelandii, as isolated under dithionite and taken at temperatures from 125 K to 175 K, are the sums of four resolved quadrupole doublets. Our results indicate that the currently accepted interpretation of these doublets can be questioned. Our data reduction method converts the M?ssbauer transmission spectra to source lineshape deconvolved absorption spectra linear in iron. We used these absorption spectra to determine the stoichiometry of the Fe clusters in MoFe-protein and we obtained much better fits if we assumed that there are four iron atoms in the 'Fe2+, doublet, two iron atoms in the 'S' doublet, twelve iron atoms in the 'D' doublet and sixteen iron atoms in the 'M' doublet. Therefore we propose that the MoFe-cofactor contains one molybdenum and eight iron atoms ('M'). We also argue that none of the previous M?ssbauer spectroscopic studies have been performed on the highest-activity preparation now obtainable, nor has there been any study to prove that the M?ssbauer spectra are independent of activity. We consider that the M?ssbauer spectroscopic studies of the MoFe-protein of nitrogenase are a re-opened and unsolved problem.     

Some contributions of I.E. Purkyn? to the visual physiology

In the research of binocular vision J. E. Purkyn? pointed out the role of retinal corresponding areas. He observed binocular interaction of pressure phosphenes elicited in them and the fusion of images projected on the corresponding foveal areas even in eyes with artificial divergency. For the first time he proposed the examination of visual fields (perimetry) in several planes and he also introduced the colour perimetry. Purkyn? analyzed profoundly the different functions of direct (foveal) and indirect (peripheral) vision. In indirect vision he anticipated the modern concept of the useful field of view. Of fundamental importance was Purkyn?'s research of the strategy and different types of ocular movements. In analysis of motion perception, he evolved the hypothesis of "general sense of space" which is very close to modern feed-back conception of "corollary discharge".