EPR spectroscopic characterization of an 'iron only' nitrogenase. S = 3/2 spectrum of component 1 isolated from Rhodobacter capsulatus.

The alternative nitrogenase of Rhodobacter capsulatus, isolated from a nifHDK deletion mutant, has been purified to near homogeneity and identified as an 'iron only' nitrogenase. The dithionite-reduced component 1 ('FeFe protein') of this enzyme showed an EPR spectrum consisting of two components: a minor S = 1/2 signal at g = 1.93 and a very characteristic S = 3/2 signal of near-stoichiometric intensity at g = 5.44. This resonance is very close to the highest possible g value (g = 5.46) for the coinciding two intradoublet subspectra of an S = 3/2 system of maximal rhombicity (E/D = 0.33). The deviation from axial symmetry (increasing E/D) correlates with the stability, activity and substrate selectivity of the different (Mo, V, Fe) nitrogenases.     

Quantitative EPR of an S = 7/2 system in thionine-oxidized MoFe proteins of nitrogenase. A redefinition of the P-cluster concept

Thionine-oxidized nitrogenase MoFe proteins from Azotobacter vinelandii. Azotobacter chroococcum and Klebsiella pneumoniae exhibit excited-state EPR signals with g = 10.4, 5.8 and 5.5 with a maximal amplitude in the temperature range of 20-50 K. The magnitude of these effective g values, combined with the temperature dependence of the peak area at g = 10.4 from 12 K to 86 K, are consistent with an S = 7/2 system with spin Hamiltonian parameters D = -3.7 +/- 0.7 cm-1, [E] = 0.16 +/- 0.01 cm-1 and g = 2.00. This interpretation predicts nine additional effective g values some of which have been detected as broad features of low intensity at g approximately 10, approximately 2.5 and approximately 1.8. The S = 7/2 EPR is ascribed to the multi-iron exchange-coupled entities known as the P clusters. Quantification relative to the S = 3/2 EPR signal from dithionite-reduced MoFe protein indicates a stoichiometry of one P cluster per FeMo cofactor. Two possible interpretations for these observations, together with data from the literature, are proposed. In the first model there are two P clusters per tetrameric MoFe protein. Each P cluster encompasses approximately 8Fe ions and releases a total of three electrons on oxidation with excess thionine. In the second model the conventional view of four P clusters, each containing approximately 4Fe, is retained. This alternative requires that following one-electron oxidation, the P clusters factorize into two populations, Pa and Pb, only one of which is further oxidized with thionine resulting in the S = 7/2 system. Both models require eight-electron oxidation of tetrameric MoFe protein to reach the S = 7/2 state.     

Q-band EPR of the S2 state of photosystem II confirms an S = 5/2 origin of the X-band g = 4.1 signal

Disagreement has remained about the spin state origin of the g = 4.1 EPR signal observed at X-band (9 GHz) from the S2 oxidation state of the Mn cluster of Photosystem II. In this study, the S2 state of PSII-enriched membrane fragments was examined at Q-band (34 GHz), with special interest in low-field signals. Light-induced signals at g = 3.1 and g = 4.6 were observed. The intensity of the signal at g = 3.1 was enhanced by the presence of F- and suppressed by the presence of 5% ethanol, indicating that it was from the same spin system as the X-band signal at g = 4.1. The Q-band signal at g = 4.6 was also enhanced by F-, but not suppressed by 5% ethanol, making its identity less clear. Although it can be accounted for by the same spin system, other sources for the signal are considered. The observation of the signal at g = 3.1 agrees well with a previous study at 15.5 GHz, in which the X-band g = 4.1 signal was proposed to arise from the middle Kramers doublet of a near rhombic S = 5/2 system. Zero-field splitting values of D = 0.455 cm(-1) and E/D = 0.25 are used to simulate the spectra.     

Factors influencing the formation of modified S2 EPR signal and the S3 EPR signal in Ca(2+)-depleted photosystem II

NaCl/EGTA-washing of photosystem II (PS-II) results in the removal of Ca2+ and the inhibition of oxygen evolution. Two new EPR signals were observed in such samples: a stable and modified S2 multiline signal and an S3 signal [(1989) Biochemistry 28, 8984-8989]. Here, we report what factors are responsible for the modifications of the S2 signal and the observation of the S3 signal. The following results were obtained. (i) The stable, modified, S2 multiline signal can be induced by the addition of high concentrations of EGTA or citrate to PS-II membranes which are already inhibited by Ca(2+)-depletion. (ii) The carboxylic acids act in the S3-state, are much less effective in S2 and have no effect in the S1-state. (iii) The extrinsic polypeptides (17- and 23-kDa) are not required to observe either the modified S2 signal or the S3 signal. However, they do influence the splitting and the lifetime of the S3 signal, and they seem to have a slight influence on the hyperfine pattern of the S2 signal. (iv) The S3 signal can be observed in Ca(2+)-depleted PS-II which does not exhibit the modified multiline signal. Then, it is proposed that formation of histidine radical during the S2 to S3 transition in Ca(2+)-depleted PS-II [(1990) Nature 347, 303-306] also occurs in functional PS-II.     

A new EPR signal associated with photosystem 1 of algal and plant photosynthesis

A new low temperature electron paramagnetic resonance (EPR) signal with a g-value of 1.97 was found in Photosystem-1 particles from a blue-green alga, Anacystis nidulans, illuminated at room temperature. A similar signal was also found in spinach Photosystem-1 particles treated with thiophenol to decrease interference from a signal due to Center A. In the dark, the signal appeared only when the Anacystis particles were at redox potentials lower than -0.5 volts where Centers A and B were also reduced. The signal is most likely due to another iron-sulfur cluster, tentatively designated as Center C. Center C could be photoreduced at low temperatures like Center A when Centers A and B were partially reduced prior to illumination, indicating possible close association of these centers in Photosystem 1 of green plant and algal photosynthesis.     

Novel EPR signals associated with FeMoco centres of MoFe protein in MgADP-inhibited turnover of nitrogenase

Two novel electron paramagnetic resonance (EPR) signals arising from the [1Mo-7Fe-9S-homocitrate] (FeMoco) centres of MoFe protein of Klebsiella pneumoniae nitrogenase (Kp1) were observed following turnover under MgATP-limited conditions. The combination of the nitrogenase Fe protein of Clostridium pasteurianum showed similar signals. The accumulation of MgADP under these conditions causes the normal EPR signal of dithionite-reduced Kp1 (with g=4.3, 3.6, 2.01) to be slowly converted to novel signals with g=4.74, 3.32, 2.00 and g=4.58, 3.50, 1.99. These signals do not form in incubation of protein mixtures containing only MgADP, thus they may be associated with trapped intermediates of the catalytic cycle.     

EPR properties of a g=2 broad signal trapped in an S1 state in Ca2+-depleted Photosystem II

We investigated a new EPR signal that gives a broad line shape around g=2 in Ca(2+)-depleted Photosystem (PS) II. The signal was trapped by illumination at 243 K in parallel with the formation of Y(Z)*. The ratio of the intensities between the g=2 broad signal and the Y(Z)* signal was 1:3, assuming a Gaussian line shape for the former. The g=2 broad signal and the Y(Z)* signal decayed together in parallel with the appearance of the S(2) state multiline at 243 K. The g=2 broad signal was assigned to be an intermediate S(1)X* state in the transition from the S(1) to the S(2) state, where X* represents an amino acid radical nearby manganese cluster, such as D1-His337. The signal is in thermal equilibrium with Y(Z)*. Possible reactions in the S state transitions in Ca(2+)-depleted PS II were discussed.     

Multifrequency EPR investigations into the origin of the S2-state signal at g = 4 of the O2-evolving complex.

The low-temperature S2-state EPR signal at g = 4 from the oxygen-evolving complex (OEC) of spinach Photosystem-II-enriched membranes is examined at three frequencies, 4 GHz (S-band), 9 GHz (X-band) and 16 GHz (P-band). While no hyperfine structure is observed at 4 GHz, the signal shows little narrowing and may mask underlying hyperfine structure. At 16 GHz, the signal shows g-anisotropy and a shift in g-components. The middle Kramers doublet of a near rhombic S = 5/2 system is found to be the only possible origin consistent with the frequency dependence of the signal. Computer simulations incorporating underlying hyperfine structure from an Mn monomer or dimer are employed to characterize the system. The low zero field splitting (ZFS) of D = 0.43 cm-1 and near rhombicity of E/D = 0.25 lead to the observed X-band g value of 4.1. Treatment with F- or NH3, which compete with Cl- for a binding site, increases the ZFS and rhombicity of the signal. These results indicate that the origin of the OEC signal at g = 4 is either an Mn(II) monomer or a coupled Mn multimer. The likelihood of a multimer is favored over that of a monomer.     

The g = 2 multiline EPR signal of the S2 state of the photosynthetic oxygen-evolving complex originates from a ground spin state.

The amplitude of the g = 2 Mn 'multiline' EPR signal of the S2 state of the photosynthetic oxygen-evolving complex varies inversely with temperature, indicating that this signal arises from a ground spin state. Electron spin echo experiments at temperatures of 4.2 K and 1.4 K show such Curie-law behavior of the g = 2 multiline EPR signal, as do continuous-wave EPR experiments performed at a non-saturating microwave power in the range from 15.0 K to 4.2 K.     

Characterization of a novel g' = 2.95 EPR signal from the binuclear center of mitochondrial cytochrome c oxidase.

The oxidized binuclear heme a3/CuB center of slow forms of bovine cytochrome oxidase exhibits a characteristic EPR signal at g' = 12. Following the (rapid) dithionite reduction of heme a and CuA, an additional EPR signal becomes apparent at g' = 2.95. As electrons enter the binuclear center this signal decays at the same slow rate as the g' = 12 signal. In the fully oxidized slow enzyme the small g' = 2.95 signal is usually masked by the g = 3 heme a signal, but it is readily detectable at low temperatures and high microwave powers. It is present in both the intrinsic and formate-ligated slow enzymes, but not in any form of fast preparation. The g' = 2.95 signal has similar temperature dependence and microwave power saturation characteristics to the g' = 12 signal. We conclude that the signal arises from the same population of binuclear centers responsible for the g' = 12 signal. The appearance of a signal at g' = 2.95 in X-band EPR is consistent with, but does not prove, the model of Hagen where the g' = 12 signal arises from a ferryl heme a3, with CuB cuprous and EPR-silent (Hagen, W. R. (1982) Biochim. Biophys. Acta 708, 82-98).     

EPR properties of a g=2 broad signal trapped in an S1 state in Ca-depleted Photosystem II

We investigated a new EPR signal that gives a broad line shape around g=2 in Ca²⁺-depleted Photosystem (PS) II. The signal was trapped by illumination at 243 K in parallel with the formation of Y_Z. The ratio of the intensities between the g=2 broad signal and the Y_Z signal was 1:3, assuming a Gaussian line shape for the former. The g=2 broad signal and the Y_Z signal decayed together in parallel with the appearance of the S₂ state multiline at 243 K. The g=2 broad signal was assigned to be an intermediate S₁X state in the transition from the S₁ to the S₂ state, where X represents an amino acid radical nearby manganese cluster, such as D1-His337. The signal is in thermal equilibrium with Y_Z. Possible reactions in the S state transitions in Ca²⁺-depleted PS II were discussed.     

The S1YZ* metalloradical EPR signal of photosystem II contains two distinct components that advance respectively to the multiline and g = 4.1 conformations of S2

The S2 state of the oxygen-evolving complex (OEC) of photosystem II is heterogeneous, exhibiting two main EPR spectral forms, the multiline and the g = 4.1 signal. It is not clearly established whether this heterogeneity develops during the S1 to S2 transition or is already present in the precursor states. We have compared the spectra of the S1YZ* intermediate, obtained by visible light excitation (induction of charge separation) of the S1 state at liquid He temperatures, (S1YZ*)vis, or by near-infrared (NIR) light excitation of the S2 state (utilization of the unusual property of the Mn cluster to act as an oxidant of Yz when excited by NIR), (S1YZ*)NIR. The decay kinetics of the (S1YZ*)vis spectrum at 11 K was also studied by the application of rapid-scan EPR. The two spectra share in common a signal with a characteristic feature at g = 2.035, but the (S1YZ*)vis spectrum contains in addition a fast decaying component 26 G wide. The analysis of the surface of the rapid-scan spectra yielded 270 +/- 35 and 90 +/- 15 s for the respective half-times of the two components of the (S1YZ*)vis spectrum at 11 K. (S1YZ*)vis advances efficiently to S2 when annealed at 200 K; notably the g = 2.035 signal advances to the multiline while the 26 G component advances to the g = 4.1 conformation. The "26 G" component is absent or very small, respectively, in thermophilic cyanobacteria or glycerol-containing spinach samples, in correlation to vanishing or very small amounts of the g = 4.1 component in the S2 spectrum. The results validate the assignment of S1YZ* to a true S1 to S2 intermediate and imply that the heterogeneity observed in S2 is already present in S1. Tentative valences are assigned to the individual Mn ions of the OEC in the two heterogeneous conformations of S1.     

The origin of the split S3 EPR signal in Ca(2+)-depleted photosystem II: histidine versus tyrosine.

The radical formed as the formal S3 charge storage state in Ca(2+)-depleted photosystem II and detected as a split EPR signal was previously assigned to an oxidized histidine radical on the basis of its UV spectrum. In a recent paper [Hallahan, B. J., Nugent, J. H. A., Warden, J. T., & Evans, M. C. W. (1992) Biochemistry 31, 4562-4573], this assignment was challenged, and it was suggested that the signal arises instead from the well-known tyrosine radical Tyrz., the electron carrier between the photooxidized chlorophyll and the Mn cluster. Here, we provide evidence that the measurements of the Tyr., on which the new interpretation was based, are artifactual due to the use of saturating microwave powers. Other than a relaxation-enhancement effect, the formation of the split S3 signal is accompanied by no change in the Tyr. signal. Although essentially unrelated to the origin of the S3 radical, several other experimental and interpretational problems in the work of Hallahan et al. (1992) are pointed out and rationalized. For example, the inability of Hallahan et al. (1992) to observe the split S3 signal in samples containing DCMU or without a chelator, in contrast to our observations, is attributed to a number of technical problems including the incomplete inhibition of the enzyme. We thus conclude that the assignment of the split S3 signal as His., although not proven, remains the most reasonable on the basis of current data.     

Electron paramagnetic resonance analysis of different Azotobacter vinelandii nitrogenase MoFe-protein conformations generated during enzyme turnover: evidence for S = 3/2 spin states from reduced MoFe-protein intermediates

Rapid-freezing experiments elicited two transient EPR signals, designated 1b and 1c, during Azotobacter vinelandii nitrogenase turnover at 23 degrees C and pH 7.4. The first of the signals to form, signal 1b, exhibited g values of 4.21 and 3.76. Its formation was at the expense of the starting EPR signal (signal 1a with g values of 4.32, 3.66, and 2.01). The second signal to arise, signal 1c, with a characteristic g value of 4.69, formed very slowly and was always of low intensity. Both signals occurred independently of the substrate being reduced. Increased electron flux through the MoFe protein caused these signals to form more rapidly. Moreover, after a MoFe-protein solution had been pretreated (using conditions of extremely low electron flux) to set up an equimolar mixture of its resting state and one-electron reduced state, these signals appeared even more rapidly when this mixture was exposed to an excess of the Fe protein. We have simulated the kinetics of formation of these EPR features using the published kinetic model for nitrogenase catalysis [Lowe, D. J., and Thorneley, R. N. F. (1984) Biochem. J. 224, 887-909] and propose that they arise from reduced states of the MoFe protein and reflect different conformations of the FeMo cofactor with different protonation states.     

Inhomogeneity of the EPR multiline signal from the S2-state of the photosystem II oxygen-evolving enzyme

The manganese complex (Mn₄) which is responsible for water oxidation in photosystem II is EPR detectable in the S₂-state, one of the five redox states of the enzyme cycle. The S₂-state is observable at 10?K either as an EPR multiline signal (spin S?=?1/2) or as a signal at g?=?4.1 (spin S?=?3/2 or 5/2). It has recently been shown that the state responsible for the multiline signal is converted to that responsible for the g?=?4.1 signal upon the absorption of near-infrared light [Boussac A, Girerd J-J, Rutherford AW (1996) Biochemistry 35?:?6984–6989]. It is shown here that the yield of the spin interconversion may be variable and depends on the photosystem II (PSII) preparations. The EPR multiline signal detected after near-infrared illumination, and which originates from PSII centers not susceptible to the near-infrared light, is shown to be different from that which originates from infrared-susceptible PSII centers. The total S₂-multiline signal results from the superposition of the two multiline signals which originate from these two PSII populations. One S₂ population gives rise to a "narrow" multiline signal characterized by strong central lines and weak outer lines. The second population gives rise to a "broad" multiline signal in which the intensity of the outer lines, at low and high field, are proportionally larger than those in the narrow multiline signal. The larger the relative amplitude of the outer lines at low and high field, the higher is the proportion of the near-infrared-susceptible PSII centers and the yield of the multiline to g?=?4.1 signal conversion. This inhomogeneity of the EPR multiline signal is briefly discussed in terms of the structural properties of the Mn₄ complex.     

A novel covalent modification of nitrogenase in a cyanobacterium

In extracts of the unicellular cyanobacterium Gloeothece, the Fe-protein of nitrogenase can be separated by SDS-PAGE into two antigenically identifiable components. Unlike the situation in photosynthetic bacteria such as Rhodospirillum rubrum, these two forms do not arise from covalent modification of the protein by ADP-ribosylation. Rather, the Fe-protein of Gloeothece nitrogenase is subjected to modification by palmitoylation.     

Detection of a g' = 1.74 EPR signal in bovine spleen purple acid phosphatase

When purified with hydroxylapatite, bovine spleen purple acid phosphatase, bearing two iron atoms/molecule, is EPR-silent. In contrast, enzyme purified without hydroxylapatite exhibits the distinctive g' = 1.74 EPR signal characteristic of porcine uteroferrin, with an intensity accounting for about 10% of the total iron. The intensity of the signal is increased 8-fold by the addition of ferrous iron. This treatment, while shifting the visible absorption maximum of the protein from 550 to 525 nm, does not significantly alter the intensity of its visible absorption. Loss of the g' = 1.74 EPR signal upon addition of phosphate to EPR-active preparations and the detection of virtually stoichiometric amounts of phosphate in the protein as isolated suggest that phosphate-binding may abolish the g' = 1.75 EPR signal. Such binding may bring the two iron atoms of the enzyme into juxtaposition, causing loss of EPR signal intensity either through spin-lattice relaxation broadening or antiferromagnetic exchange coupling, perhaps involving phosphate or other ligands intercalated between the two paramagnetic iron atoms.