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P J Stephens

Publications and source records attributed to P J Stephens.

At least 73 records · Page 4Linked to original sources
PubMed1985

[4Fe-4S]-cluster-depleted Azotobacter vinelandii ferredoxin I: a new 3Fe iron-sulfur protein.

Fe(CN)6(-3) oxidation of the aerobically isolated 7Fe Azotobacter vinelandii ferredoxin I, (7Fe)FdI, is a degradative reaction. Destruction of the [4Fe-4S] cluster occurs first, followed by destruction of the [3Fe-3S] cluster. At a Fe(CN)6(-3)/(7Fe)FdI concentration ratio of 20, the product is a mixture of apoprotein and protein containing only a [3Fe-3S] cluster, (3Fe)FdI. This protein mixture, after partial purification, has been characterized by absorption, CD, magnetic CD, and EPR and Fe x-ray absorption spectroscopies. EPR and magnetic CD spectra provide strong evidence that the [3Fe-3S] cluster in (3Fe)FdI is essentially identical in structure to that in (7Fe)FdI. Analysis of the extended x-ray absorption fine structure (EXAFS) of (3Fe)FdI finds Fe scattering at an average Fe...Fe distance of approximately equal to 2.7 A. The structure of the oxidized [3Fe-3S] cluster in solutions of oxidized (3Fe)FdI, and, by extension, of oxidized (7Fe)FdI, is thus different from that obtained by x-ray crystallography on oxidized (7Fe)FdI. Possible interpretations of this result are discussed.

Azotobacter↗
PubMed1985

Polyneuronal innervation of an adult and embryonic lobster muscle.

Motor innervation of the deep extensor muscle in the abdomen of lobsters (Homarus americanus) was compared in adults and embryos using electrophysiological techniques. There is widespread innervation of the adult muscle by the common excitor and inhibitor axons and regionally restricted or private innervation by three more excitor axons. In the embryo the earliest sign of functional innervation revealed a single inhibitory and two to three excitatory axons thus denoting simultaneous innervation by the full complement of axons. In corroboration, serial-section electron microscopy revealed several axon profiles invading the embryonic deep extensor muscles and giving rise to well-defined neuromuscular synapses with presynaptic dense bars. Innervation patterns to homologous regions of the embryonic and adult muscles were similar, consisting of a few large inhibitory synapses and many small excitatory ones. Consequently the adult pattern of polyneuronal innervation occurs simultaneously and in toto during embryonic development.

Animals↗
PubMed1984

Neuromuscular relationships during claw regeneration in Californian snapping shrimp.

We have examined the innervation patterns of the two excitor axons to the closer muscle in the dimorphic (snapper and pincer) claws of Californian snapping shrimp (Alpheus californiensis). In both claws the fast-closer excitor (FCE) axon supplies all of the closer muscle fibers. The slow-closer excitor (SCE) axon, however, makes functional connections only with fibers on the dorsal and ventral margins, which are composed of slow-type fibers in the large snapper claw and of intermediate-type fibers in the smaller pincer claw. The central band of fast fibers in the pincer closer muscle is not innervated by the SCE. During claw regeneration, the closer muscle is initially composed of a population of fast fibers in the pincer and intermediate-type fibers in the snapper. The innervation patterns of the two excitatory motor axons in regenerating claws at this stage are the same as in fully developed claws. During the first intermolt period some fast-closer muscle fibers in the pincer claw differentiate into intermediate-type fibers, but the axon innervation patterns do not change. If the correlation between SCE axon innervation pattern and the regional distribution of different muscle fiber types is indicative of nerve-muscle interactions, the present data suggest that the trophic influence must proceed from nerve to muscle.

Animals↗
PubMed1984

Spectroscopic studies of ferricyanide oxidation of Azotobacter vinelandii ferredoxin I.

The Fe(CN)3-(6) oxidation of the crystallographically characterized [[3Fe-3S], [4Fe-4S]] ferredoxin I of Azotobacter vinelandii has been studied using absorption, circular dichroism, magnetic circular dichroism, and EPR spectroscopies. A paramagnetic intermediate is observed en route to Fe-S cluster-free apoprotein, possessing an anisotropic g approximately equal to 2 EPR signal, surviving to temperatures greater than 77 K. This species is shown to result from 3-electron oxidation of the [4Fe-4S] cluster, without modification of the [3Fe-3S] cluster. However, it does not give rise to observable paramagnetic magnetic circular dichroism in the visible-near UV spectral region and is therefore neither an oxidized HIPIP [4Fe-4S] cluster nor an oxidized [3Fe-3S] cluster. We identify the paramagnetic species as a cysteinyldisulfide radical formed on dissociation of an oxidized cysteinate and an oxidized sulfide ion from the [4Fe-4S] cluster. This conclusion is consistent with the observed reaction stoichiometry, the spectroscopic results obtained, known EPR spectra of disulfide radicals, and the reconstitution of the native [4Fe-4S] cluster by dithiothreitol alone. This reaction, earlier interpreted as a HIPIP-type oxidation, is a previously uncharacterized oxidation reaction of [4Fe-4S] clusters.

Azotobacter↗
PubMed1982

The peripheral generation of action potentials in excitatory motor neurons of a crab.

The effects of temperature on the firing patterns of the excitatory motor supply to the bender muscle were examined in autotomized walking limbs of the crab Pachygrapsus crassipes. A brief electrical shock applied to the limb nerve evoked an action potential in one or both of the excitor axons and an excitatory junctional potential (ejp) in the bender muscle fibers. However, at high temperatures a single shock to one of the excitor axons (E2) resulted in a short train of nerve spikes and a concomitant burst of ejps. Extracellular recordings made from two sites along the E2 axon demonstrated that the additional action potentials are generated peripherally. Furthermore, myogram recordings from bender muscles in untethered animals indicate that peripheral generation of spikes does take place in intact crabs exposed to high temperatures. The peripheral generation of action potentials exhibited a critical temperature threshold which could be altered by acclimation and by low levels of ethanol in the saline bathing the autotomized limb preparation. This phenomenon may, therefore, be closely associated with membrane fluidity.

Action Potentials↗
PubMed1982

Thermal acclimation in a crustacean neuromuscular system.

1. Effects of temperature on the muscle fibre membrane and synapses of stretcher muscle preparations made from autotomized limbs of the Pacific shore crab (Pachygrapsus crassipes) were investigated. 2. Acclimation of the crabs to different temperatures modified properties of both muscle fibre membrane and synapses. 3. Increased temperature produced an increase in membrane potential of the muscle fibres. A semi-log plot of these data revealed two linear phases of the membrane potential-temperature relationship, with a change in slope near the acclimation temperature. 4. Maximum values for excitatory junction potential (EJP) amplitude and time constant of EJP decay, and minimum values for facilitation were obtained at temperatures close to the acclimation temperature. It is suggested that the decline in EJP amplitude and time constant of decay produced by deviations in temperature from the acclimation temperature is compensated for by an increase in the amount of facilitation. In this way, maximum tension can be produced by the stretcher muscle in a range of at least 8 degrees C around the acclimation temperature.

Adaptation, Physiological↗
PubMed1981

Ethanol and temperature modify motor axon firing patterns.

At high temperature a single orthodromic action potential in the excitor (E) axon to the stretcher muscle of the crab Pachygrapsus crassipes provokes the generation of additional spikes in the peripheral E axon branches. This phenomenon exhibits a critical temperature threshold. Bathing preparations in crab salines made up with ethanol at levels as low as 0.5% (109 mM) reversibly decreased the temperature threshold for peripheral spike generation. Higher levels of ethanol resulted in the peripheral generation of E axon spikes at lower temperatures. These results, together with the observation that the temperature threshold is dependent upon the acclimation temperature, indicate that the peripheral generation of action potentials in the E axon is associated with an increase membrane lipid fluidity.

Animals↗
PubMed1981

Circular dichroism and magnetic circular dichroism of reduced molybdenum-iron protein of Azotobacter vinelandii nitrogenase.

Studies of the circular dichroism (CD) and magnetic circular dichroism (MCD) of the dithionite-reduced molybdenum-iron protein of Azotobacter vinelandii nitrogenase (Av1) are reported. CD and MCD are measurable at room temperature across a wide spectral range, from the near-UV to the near-IR. The visible-near-UV CD is insignificantly affected by moderate variations in pH, temperature, ionic strength, and buffer, providing evidence against conformational change in the range studied. Mg2+ and ATP also cause no observable change in the visible-near-UV CD. Both CD and MCD in the visible-near-UV are unaffected by 30% inactivation by O2. However, the CD and MCD spectra of uncrystallized Av1 differ very significantly from those of crystallized Av1; in particular, the MCD spectrum is very sensitive to the presence of heme impurities. The identicality in both CD and MCD spectra of the reduced molybdenum-iron proteins from Azotobacter vinelandii and Klebsiella pneumoniae shows that these proteins contain metal clusters, identical in number, structure, and protein environment. While the absorption, CD, and MCD spectra of reduced Av1 are typical in many respects of simpler iron-sulfur proteins and are most similar to the [Fe4S4(SR)4]3- clusters found in reduced bacterial ferredoxins, significant differences exist. It is concluded, therefore, that the clusters present are not identical with those previously characterized, a conclusion earlier arrived at from electron paramagnetic resonance, Mössbauer, and EXAFS spectroscopies.

Azotobacter↗
PubMed1981

Spectroscopic studies of ascorbate oxidase. Electronic structure of the blue copper sites.

Low-temperature electronic absorption and room temperature circular dichroism (CD) and magnetic circular dichroism (MCD) spectra are reported for ascorbate oxidase. Bands attributable to d-d electronic transitions in the type 1 (blue) coppers (2B2 ground state) have been observed at 5800, 10000, and 12000 cm-1 (1725, 1000, and 835 nm). The three bands are assigned to the transitions 2B2 leads to 2E, 2B2 leads to 2B1, and 2B2 leads to 2A1, respectively, in the slightly flattened tetrahedral blue copper centers. The ligand field theoretical treatment predicts a type 1 copper reduction potential of 417 mV. The MCD spectrum of ascorbate oxidase contains one major band at 14000 cm-1 (710 nm) and a very weak feature at 13 200 cm-1 (550 nm). The intensities of the CD and MCD bands are consistent with two or three type 1 coppers in each enzyme molecule, and the near-infrared spectroscopic results suggest that the electronic structures of the blue coppers are closely similar.

Ascorbate Oxidase↗
PubMed1979

Circular dichroism and magnetic circular dichroism of nitrogenase proteins.

Circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of nitrogenase components (MoFe protein and Fe protein) from Azotobacter vinelandii (Av) and Klebsiella pneumoniae (Kp) have been obtained in the near infrared-visible-near ultraviolet spectral region. Previously, visible CD was reported to be absent or barely detectable in nitrogenase proteins; MCD spectra have not been reported. The chiroptical spectra can be measured in solution at room temperature, an advantage relative to spectroscopic methods requiring cryogenic sample temperatures. Absorption spectra were also obtained. The CD and MCD are markedly more structured, and thus interpretively more useful, than the corresponding absorption spectra. The dithionite-reduced MoFe proteins (Av1, Kp1) have nearly identical CD and MCD, demonstrating identical numbers and types of metal centers in similar protein environments. The CD and MCD cannot be explained solely in terms of contributions from known 4-Fe or 2-Fe clusters; the near-infrared MCD is inconsistent with the presence of known 4-Fe clusters. CD and MCD spectra of Lauth's violet-oxidized Kp1 are also reported. The reduced Fe proteins (Av2, Kp2) have similar CD and MCD, again indicating significant conservation of chromophore environment. The spectra clearly demonstrate the presence of a reduced bacterial ferredoxin-like (C(3-)) 4-Fe cluster. No obvious evidence of additional chromophores is observed. CD, MCD, and absorption spectra of Av1-oxidized Av2 are reported. The absorption spectrum shows the expected shoulder near 390 nm. The CD and MCD are characteristic of a C(2-) 4-Fe cluster; in particular, the diagnostic near-infrared MCD peak is observed at approximately 8300 cm(-1). The CD of Av2 oxidized in the presence and absence of MgATP are radically different, providing the first direct evidence for MgATP interaction with Fe protein in this oxidation state.

Azotobacter↗
PubMed1978

Circular dichroism and magnetic circular dichroism of iron-sulfur proteins.

Circular dichroism (CD) and magnetic circular dichroism (MCD) spectra are reported for the 2-Fe ferredoxins from Pseudomonas putida and Spirulina maxima, Chromatium HIPIP, the 4-Fe ferredoxin from Bacillus stearothermophilus, and the 8-Fe ferredoxin from Clostridium pasteurianum. The spectral range spans the near-infrared, visible, and near ultraviolet. In all cases except oxidized 2-Fe ferredoxins, electronic absorption is observed continuously from less than 5000 cm-1 to above 30,000 cm-1. The CD spectra of the two 2-Fe ferredoxins are similar. In contrast, the CD of the 4-Fe and 8-Fe proteins, for a given 4-Fe cluster oxidation level, varies considerable with protein. MCD is less sensitive to protein environment than is CD. In the 2-Fe proteins, MCD at 5 T is appreciably smaller than the CD; in the 4-Fe and 8-Fe proteins, MCD and CD are comparable in magnitude. Both CD and MCD are more highly structured than the corresponding absorption spectra. The CD and MCD spectra reported provide a broader base than heretofore available for the characterization of iron-sulfur proteins containing 2-Fe and 4-Fe clusters and for the evaluation of electronic structural models for these clusters.

Bacteria↗
PubMed1978

Cluster characterization in iron-sulfur proteins by magnetic circular dichroism.

We report magnetic circular dichroism (MCD) spectra of 4-Fe iron-sulfur clusters in the iron-sulfur proteins Chromatium high-potential iron protein (HIPIP), Bacillus stearothermophilus ferredoxin and Clostridium pasteurianum ferredoxin. The MCD is found to vary significantly with cluster oxidation state but is relatively insensitive to the nature of the protein. The spectra obtained are compared with the corresponding spectra of iron-sulfur proteins containing 2-Fe clusters. It is concluded that MCD is useful for the characterization of iron-sulfur cluster type and oxidation state in iron-sulfur proteins and is superior for this purpose to absorption and natural circular dichroism spectroscopy.

Chromatium↗
PubMed1977

Near-infrared magnetic circular dichroism of cytochrome c'.

The near-infrared magnetic circular dichroism (MCD) of Rhodospirillum rubrum, Chromatium vinosum, and Rhodopseudomonas palustris cytochromes c' are reported. The spectra of the reduced protein are very similar to those of deoxymyoglobin. The spectra of the oxidized proteins in the pD range 1-13 can be analyzed on the basis of four species A, B, C, and D. The existence of nine species, reported in a recent electron paramagnetic resonance study, is not substantiated. The MCD spectra support the assignment of B as high spin and C and D as low spin. The MCD of species A is close to that of high-spin proteins and does not support the recently proposed assignment of a mixed high- and intermediate-spin ground state for this species. The energies of the near-IR electronic transitions of all four oxidized species point to axial ligation via oxygen, assuming histidine to be the opposite axial ligand. Unfortunately, insufficient model compounds with ligation by carboxyl or hydroxyl moieties exist to enable more precise assignments.

Chromatium↗