Brian J. Hales

Immobilized radicals. IV. Biological semiquinone anions and neutral semiquinones

Abstract The semiquinone anion and neutral semiquinone radicals of benzoquinone, vitamin K-1, ubiquinone and plastoquinone were generated in both protic and aprotic solvents and frozen to produce immobilized spectra. The linewidths of the neutral semiquinones were always much larger than those of the corresponding anion radicals. Furthermore, the spectra of the neutral radicals often exhibit fine structure. When compared with in vivo spectra of semiquinones, these model systems suggest that the ubisemiquinone anion radical observed in photosynthetic bacteria can exist in either a protic or aprotic environment. There is also the implication that Signal II in chloroplasts may be a plastosemiquinone radical with a spin distribution similar to that of the neutral radical.

Supposition of the origin of Signal II from random and oriented chloroplasts

Abstract From previous studies of biological semiquinones in different solvents, the origin of Signal II in chloroplasts is hypothesized to be a plastosemiquinone anion radical perturbed by a metal cation. Assuming this model, theoretical principal g factors and hyperfine splitting constants were calculated and used to simulate the random spectrum of spinach Signal II. Oriented chloroplasts were used to determine the principal angles of this model. Oriented chloroplasts from collard greens showed a different angular dependency of Signal II from those of spinach as well as the presence of added fine structure.

Variable temperature magnetic circular dichroism studies of reduced nitrogenase iron proteins and [4Fe-4S]+ synthetic analog clusters

Variable temperature magnetic circular dichroism (VTMCD) and EPR spectroscopies have been used to investigate the ground and excited-state properties of [4Fe-4S]+ clusters in Mo- and V-nitrogenase Fe-proteins from Azotobacter vinelandii and two synthetic analog clusters, [Fe4S4(SEt)4]3- and [Fe4S4(SC6H11)4]3-. The results indicate similar [4Fe-4S]+ clusters with analogous S = 1/2 and S = 3/2 ground states in both Fe-proteins. However, the Fe-proteins do differ in terms of the medium effects on the S = 1/2 and S = 3/2 spin mixtures in frozen solution. By utilizing medium effects in both Fe-proteins, the VTMCD characteristics of both the S = 1/2 and S = 3/2 forms of the [4Fe-4S]+ have been determined. Together with the VTMCD studies of [Fe4S4(SEt)4]3- and [Fe4S4(SC6H11)4]3-, which are shown to be predominantly S = 1/2 and 3/2, respectively, in frozen DMF/toluene solutions, the results demonstrate that the form of the VTMCD spectra provides a means of identifying and distinguishing S = 1/2 and S = 3/2 [4Fe-4S]+ clusters. Ground state zero-field splitting parameters for the S = 3/2 clusters are determined for both Fe-proteins. In addition to spin state heterogeneity, samples of the Mo-nitrogenase Fe-protein in the presence of 50% (v/v) ethylene glycol were found to exhibit heterogeneity in the S = 1/2 resonance. A rapidly relaxing axial resonance, g perpendicular = 1.94 and g parallel = 1.82, was observed in addition to the characteristic rhombic resonance, g = 2.05, 1.94 and 1.87. The origin of the heterogeneity exhibited by [4Fe-4S]+ clusters in frozen solution is discussed in light of these results.

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.

Variant MoFe proteins of Azotobacter vinelandii: effects of carbon monoxide on electron paramagnetic resonance spectra generated during enzyme turnover.

The resting state of wild-type nitrogenase MoFe protein exhibits an S=3/2 electron paramagnetic resonance (EPR) signal originating from the FeMo cofactor, the enzyme’s active site. When nitrogenase turns over under CO, this signal disappears and one (sometimes two) of three new EPR signals, which also arise from the FeMo cofactor, appears, depending on the CO concentration. The appearance and properties of these CO-inducible EPR signals, which were also generated with variant MoFe proteins (αR96Q, αR96K, αQ191K, αR359K, αR96K/αR359K, αR277C, αR277H, and ΔnifV) that are impacted around the FeMo cofactor, have been investigated. No new CO-induced EPR signals arise from any variant, suggesting that no new CO-binding sites are produced by the substitutions. All variant proteins, except αR277H, produce the lo-CO signal; all, except αQ191K, produce the hi(5)-CO signal; but only two (αR96Q and ΔnifV) exhibit the hi-CO signal. FeMo cofactor’s environment clearly dictates which CO-induced EPR signals are generated; however, none of these EPR signals correlate with CO inhibition of H2 evolution observed with some of these variants. CO inhibition of H2 evolution is, therefore, due to CO binding to a different site(s) from those responsible for the CO-induced EPR signals. Some resting-state variants have overlapping S=3/2 EPR signals, whose intensities simultaneously decrease under turnover conditions, indicating that all FeMo cofactor conformations are catalytically active. Moreover, these variants produce a similar number of hi(5)-CO signals after turnover under CO to the number of resting-state S=3/2 signals. The FeMo cofactor associated with the hi(5)-CO signal likely contains two bridging CO molecules.

Immobilized radicals. III. Anisotropic saturation of semiquinones in protic solvents

The nonuniform saturation behavior of semiquinones was investigated as a function of temperature, solvent, and ESR microwave frequency levels. Results from these studies show that this effect occurs only in protic solvents at temperatures equal to and below the solvents’ freezing point. This phenomenon is theorized to be due to the strong hydrogen bonds experienced by semiquinones in these solvents. Apparently, the radical’s relaxation behavior along the direction of the hydrogen bonds is significantly different from the relaxation along the remaining two principal axes. The nonuniform saturation behavior is, therefore, an anisotropic relaxation phenomenon.

Photo-lability of CO bound to Mo-nitrogenase from Azotobacter vinelandii

In the presence of CO and under turnover conditions, Mo-nitrogenase generates three different electron paramagnetic resonance (EPR) signals. One of the signals, lo-CO, is an S=1/2 signal and occurs under low CO concentrations. The other two signals, hi-CO (S=1/2) and hi(5)-CO (S=3/2) displace the lo-CO as the CO concentration is raised above 0.05 atm. Irradiation of hi-CO with visible light at 12 K converts it into lo-CO. Using a series of color filters, the corrected action spectrum is determined and shown to contain 2-3 broad maxima in the region 350-730 nm. The conversion of lo-CO back into hi-CO is accomplished by warming the sample to 77 K for 5 min. Using this temperature cycle, the rate constant for the re-association of CO with lo-CO to form hi-CO is determined in the range 12-90 K. From these data, the activation energy for this reaction is calculated to be 3.9 kJ/mol. Identical irradiation of either lo-CO or hi(5)-CO induces no spectral change, showing that both of these states are photo-stable. The photo-stability of hi(5)-CO demonstrates that it is structurally different from hi-CO.

VTVH-MCD study of the Delta nifB Delta nifZ MoFe protein from Azotobacter vinelandii.

NifZ is a member of a series of proteins associated with the maturation of the nitrogenase MoFe protein. An MCD spectroscopic study was undertaken on the Delta nifB Delta nifZ MoFe protein generated in the absence of both NifZ and NifB (deletion of NifB generates an apo-MoFe protein lacking the FeMo cofactor). Results presented here show that, in the absence of NifZ, only one of the two P-clusters of the MoFe protein is matured to the ultimate [8Fe-7S] structure. The other P-cluster site in the protein contains a [4Fe-4S] cluster pair, representing a P-cluster precursor that is electronically identical to the analogous clusters observed in the Delta nifH MoFe protein. These results suggest that the MoFe protein is synthesized in a stepwise fashion where NifZ is specifically required for the formation of the second P-cluster.

(4Fe4S) 2+ Clusters Exhibit Ground-State Paramagnetism

Two proteins involved in nitrogen fixation contain ferredoxin-type [4Fe4S] clusters that exist in paramagnetic ground state upon oxidation, a property never observed since the discovery of ferredoxins 50 years ago. This unique characteristic suggests a specific coupling in these clusters necessary for nitrogen fixation and implies an evolutionary connection between the clusters in the two proteins.

P+ state of nitrogenase p-cluster exhibits electronic structure of a [Fe4S4]+ cluster.

Mo nitrogenase consists of two component proteins: the Fe protein, which contains a [Fe(4)S(4)] cluster, and the MoFe protein, which contains two different classes of metal cluster: P-cluster ([Fe(8)S(7)]) and FeMoco ([MoFe(7)S(9)C·homocitrate]). The P-cluster is believed to mediate the electron transfer between the Fe protein and the MoFe protein via interconversions between its various oxidation states, such as the all-ferrous state (P(N)) and the one- (P(+)) and two-electron (P(2+)) oxidized states. While the structural and electronic properties of P(N) and P(2+) states have been well characterized, little is known about the electronic structure of the P(+) state. Here, a mutant strain of Azotobacter vinelandii (DJ1193) was used to facilitate the characterization of the P(+) state of P-cluster. This strain expresses a MoFe protein variant (designated ΔnifB β-188(Cys) MoFe protein) that accumulates the P(+) form of P-cluster in the resting state. Magnetic circular dichroism (MCD) spectrum of the P-cluster in the oxidized ΔnifB β-188(Cys) MoFe protein closely resembles that of the P(2+) state in the oxidized wild-type MoFe protein, except for the absence of a major charge-transfer band centered at 823 nm. Moreover, magnetization curves of ΔnifB β-188(Cys) and wild-type MoFe proteins suggest that the P(2+) species in both proteins have the same spin state. MCD spectrum of the P(+) state in the ΔnifB β-188(Cys) MoFe protein, on the other hand, is associated with a classic [Fe(4)S(4)](+) cluster, suggesting that the P-cluster could be viewed as two coupled 4Fe clusters and that it could donate either one or two electrons to FeMoco by using one or both of its 4Fe halves. Such a mode of action of P-cluster could provide energetic and kinetic advantages to nitrogenase in the complex mechanism of N(2) reduction.