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B J Hales

Publications and source records attributed to B J Hales.

12 recordsLinked to original sources
PubMed1990

Alternative nitrogenase.

Recently, it has been demonstrated that both A. vinelandii and A. chroococcum have the ability to synthesize several different nitrogen-fixing enzymes. Both species can produce a Mo- or V-containing nitrogenase while A. vinelandii can also generate an all-Fe form of the enzyme. Regardless of the source or form of the enzyme, all nitrogenases are composed of two separable proteins, called components 1 and 2, where component 2 is a highly conserved dimer containing a single [4Fe-4S] cluster. The major differences among the various forms of the enzyme are in component 1, the protein where substrate reduction occurs. This protein can exist in forms of four, five, or six subunits and can contain Mo, V or only Fe at the putative active site. Finally, there are also variations among the different enzyme systems regarding the paramagnetism of component 1 as well as the general substrate reduction patterns. While it is not yet known why these bacteria possess the ability to generate multiple forms of nitrogenase or which form of the enzyme has the greatest physiological importance, the existence of these various nitrogenases raises several other important questions. Specifically, how do Mo, V and Fe regulate the synthesis of each enzyme system, which genes are unique and which are common for these systems, and what roles, if any, do Mo, V and Fe play in catalysis? The fact that we can now use three different enzyme systems to investigate nitrogen fixation may greatly increase our ability to answer these questions and eventually understand the mechanism of this very important enzymatic reaction.

Azotobacter↗
PubMed1987

Nitrogen fixation by Azotobacter vinelandii in tungsten-containing medium.

Nitrogenase was isolated and purified from wild-type and a tungsten-resistant mutant (LM2) of Azotobacter vinelandii strain OP derepressed on medium containing 1-10 mM W. While the enzyme from the wild-type strain contained the polypeptides of the conventional enzyme, metal analysis of component 1 demonstrated the existence of one atom each of molybdenum and tungsten. Furthermore, the ESR spectrum of this protein contained three signals, two of which originated from S = 3/2 spin states. One of these signals is nearly identical to that of the conventional MoFe-protein while the other is hypothesized to originate from a W-containing cofactor. In spite of the presence of W, the substrate reduction pattern of this enzyme is the same as that of the conventional enzyme.

Azotobacter↗
PubMed1987

Characterization of the metal clusters in the nitrogenase molybdenum-iron and vanadium-iron proteins of Azotobacter vinelandii using magnetic circular dichroism spectroscopy.

Low-temperature magnetic circular dichroism (MCD) spectroscopy has been used to investigate the metal clusters in the conventional nitrogenase MoFe protein and alternative VFe protein from Azotobacter vinelandii. In the dithionite-reduced state, the MCD spectrum of the MoFe protein is extremely similar to that previously observed for the S = 3/2 spin state of the M clusters in the MoFe protein of Klebsiella pneumoniae. A paramagnetic cluster with an S = 3/2 ground state is also responsible for the temperature-dependent MCD transitions of dithionite-reduced VFe protein. However, the electronic and magnetic properties of this cluster are quite distinct from those of M centers in conventional nitrogenase. When these proteins are oxidized with thionine, the MoFe protein exhibits MCD spectra and magnetization characteristics identical with those observed for the P clusters in K. pneumoniae, while those of the VFe protein are only similar. However, the paramagnetism in the thionine-oxidized VFe protein, like the conventional enzyme, probably arises from an S = 5/2 spin system with near-axial symmetry and a negative zero-field splitting. Novel clusters with electronic, magnetic, and redox properties similar to those of conventional P clusters are, therefore, present in the VFe protein.

Azotobacter↗
PubMed1986

Isolation of a new vanadium-containing nitrogenase from Azotobacter vinelandii.

A new nitrogenase from Azotobacter vinelandii has been isolated and characterized. It consists of two proteins, one of which is almost identical with the Fe protein (component 2) of the conventional enzyme. The second protein (Av1'), however, has now been isolated and shown to differ completely from conventional component 1, i.e., the MoFe protein. This new protein consists of two polypeptides with a total molecular weight of around 200,000. In place of Mo and Fe it contains V and Fe with a V:Fe ratio of 1:13 +/- 3. The ESR spectrum of Av1' also differs from conventional component 1 in that lacks the g = 3.6 resonance that arises from the FeMo cofactor but contains an axial signal with gav less than 2 as well as inflections in the g = 4-6 region possibly arising from an S = 3/2 state. This new enzyme can reduce dinitrogen, protons, and acetylene but is only able to utilize 10-15% of its electrons for the reduction of acetylene.

Azotobacter↗
PubMed1986

Isolation and characterization of a second nitrogenase Fe-protein from Azotobacter vinelandii.

Wild-type Azotobacter vinelandii strain UW was transformed with plasmid pDB12 to produce a species (LS10) unable to synthesize the structural proteins of component 1 and component 2 of native nitrogenase. A spontaneous mutant of this strain was isolated (LS15) which can grow by nitrogen fixation in the presence or absence of either Mo or W. It is proposed that LS15 fixes nitrogen solely by an alternative nitrogen-fixing system which previously has been hypothesized to exist in A. vinelandii. Under nitrogen-fixing conditions, LS15 synthesizes a protein similar to component 2 (Av2) of native nitrogenase in that it can complement native component 1 (Av1) for enzymatic activity. Isolation and characterization of this second component 2 shows it to be a 4Fe-4S protein of molecular mass about 62 kDa and is antigenically similar to Av2. This protein is also similar to Av2 in that in the reduced state it possesses a rhombic ESR spectrum in the g = 2 region, which changes to an axial spectrum upon addition of MgATP. It is suggested that this second Fe-protein is associated with the alternative nitrogen-fixing system in A. vinelandii.

Azotobacter↗
PubMed1985

In vivo interaction between nitrogenase molybdenum-iron protein and membrane in Azotobacter vinelandii and Rhodospirillum rubrum.

Oriented whole cell multilayers of Azotobacter vinelandii and Rhodospirillum rubrum were analyzed by electron spin resonance (ESR) spectroscopy to detect possible structural associations between nitrogenase molybdenum-iron (MoFe) protein and cytoplasmic or intracytoplasmic membrane. Initially, protocols were designed to obtain strong molybdenum-iron protein ESR signals in whole cell samples of each organism. Then, two-dimensional orientation of whole cell membranes was demonstrated in whole cell multilayers using doxyl stearate spin label in A. vinelandii and the bacteriochlorophyll a dimer triplet signal, (BCHl a)T2, from the intracytoplasmic membrane-bound photosynthetic apparatus of R. rubrum. Subsequent analysis of the low-field signals, g = 4.3 and g = 3.6, of molybdenum-iron protein in whole cell multilayers of each organism showed orientation-dependent characteristics, although the properties of each were different. Specifically, as the normal to the membrane plane was rotated from perpendicular to parallel with the ESR magnetic field, the amplitude of the g = 3.6 signal decreased from maximum to about 37% of maximum in A. vinelandii and from maximum to about 88% of maximum in R. rubrum. The angular dependence of the g = 4.3 peak during rotation varied in A. vinelandii, but decreased from maximum to about 63% of maximum in R. rubrum. These data suggest that the molybdenum-iron protein of nitrogenase was oriented in response to the physical orientation of cellular membranes and that a structural association may exist between this nitrogenase component and membrane in these organisms.

Azotobacter↗
PubMed1979

Orientation of the bacteriochlorophyll triplet and the primary ubiquinone acceptor of Rhodospirillum rubrum in membrane multilayers determined by ESR spectroscopy (I).

Chromatophores from Rhodospirillum rubrum were oriented as multilayers on quartz slides under reducing conditions. Irradiation of these multilayers in the resonance cavity of an ESR spectrometer at 6 K yielded the spectrum of the bacteriochlorophyll dimer triplet. The relative intesities of the main six lines of the triplet were dependent on the angle subtended by the direction of the external magnetic field with plane of the multilayers. The angular dependence of the intensities of these transitions can best be interpreted in terms of one of the principal axes of the triplet lying along the plane of the membrane while the other two axes are titled 10--20 degrees away from the parallel to and normal to the membrane directions. If we assume the porphyrin planes of the dimer to be parallel and the largest splitting of the triplet transitions to correspond to those transitions in a direction normal to this plane, then these data imply that the dimer planes are nearly perpendicular to the membrane plane. Purified iron-depleted phototrap complexes were similarly oriented in reconstituted phosphatidylcholine multilayers and the angular dependence of the light-induced spectrum recorded at room temperature. A computer analysis of this angular dependence suggests that the plane of the primary ubiquinone acceptor molecule is parallel to the plane of the membrane and therefore perpendicular to the donor.

Bacterial Chromatophores↗
PubMed1976

Temperature dependency of the rate of electron transport as a monitor of protein motion.

The temperature dependency of the rate of biological electron transport is interpreted as evolving from a contraction of the electron transport components. A theoretical expression for this temperature dependency is derived in terms of the coefficient of linear expansion (a) of the protein components. Using this expression alpha is calculated for several electron transport systems and shown to be similar to alpha-values of synthetic polymers. A discontinuity in alpha is shown to be present in all biological electron transport reactions at ca. 150 K. This discontinuity is interpreted as a change in the intramolecular bonding of the electron transport protein units.

Bacterial Proteins↗