Studies on the iron - sulfur clusters of hydrogenase, sulfite reductase, nitrogenase and the prismane protein

Iron-sulfur clusters are present in a large number of proteins. Sofar structures of four types of protein-bound iron-sulfur clusters have been determined by X-ray diffraction: rubredoxin-like, [2Fe-2S], [3Fe-4S] and [4Fe-4S] centers. The presence of any of these clusters in a protein can be predicted by comparison of spectroscopic properties. However a number of multiple-electron transferring enzymes, like the Fe-only hydrogenase, sulfite reductase and nitrogenase MoFe protein have enigmatic iron-sulfur clusters with spectroscopic properties unlike those of the known structures. These enzymes share a high iron and acidlabile sulfur content and the presence of superspin systems with S≥5/2. In this thesis biochemical and spectroscopic studies are presented on the above-mentioned iron-sulfur proteins and two unusual newly discovered iron-sulfur proteins, the 'prismane' protein and nigerythrin.Chapter 2 summarizes new findings on the Fe-only hydrogenase and the redox properties of its cubanes and hydrogen activating iron-sulfur H-cluster. The hydrogenase, aerobically isolated from the sulfate-reducing bacterium <em>Desulfovibrio vulgaris</em> (Hildenborough) was shown to be composed of a mixture of high and low activity charge conformers, which can be isolated and discriminated by chromatographic and electrophoretic techniques. The redox properties of the rhombic S=1/2 EPR signals associated with the H-cluster of hydrogenase preactivated with dithionite or hydrogen were considerably more simple than those reported by Patil and coworkers for the (oxygen-insenstive) resting enzyme. Instead of sequential bell-shaped curves for the rhombic g=2.07 (g=1.96 and g=1.89) and g=2.11 (g=2.05 and g=2.00) EPR signals in reductive dye-mediated titrations, a simple behaviour with two redox states was observed both in reductive and oxidative titrations. The interconversion between the diamagnetic reduced and the oxidized redox state of the H-cluster exhibiting the g=2.11 S=1/2 EPR signal occurred at -307 mV. The bell-shaped nature and the occurrence of the g=2.07 rhombic EPR signal thus was due to the activation process of the H-cluster. By equilibration with H ₂ /H <sup>+</SUP>of activated hydrogenase a midpoint potential of -330 mV was determined for the cubanes. A similar midpoint potential was observed in a dyemediated titration of a recombinant hydrogenase lacking the H-cluster. In these experiments no evidence for redox interaction between the two cubanes was seen.In the course of extensive purification procedures of the Fe-hydrogenase it was recognized that an iron-sulfur protein with novel EPR spectroscopic properties occasionally contaminated hydrogenase preparations (Chapter 3). The as isolated form had a substoichiometric S=1/2 EPR signal with g=1.97, g=1.95 and g=1.90. Chemical analysis showed that, although such g-values are typical for Mo <sup>5+</SUP>= (or W <sup>5+</SUP>) no metals other than iron were present. The 'molybdenum'-like EPR spectrum disappeared both on reduction and oxidation. In the dithionite reduced form an almost stoichiometric S=1/2 EPR signal was observed. The g-values (g=2.00, 1.82 and 1.32) were reminiscent of those of prismane model compounds in the [6Fe-6S] <sup>3+</SUP>redox state. Therefore the protein was proposed to be a prismane-containing protein, in agreement with the chemical analysis indicating ≈6 Fe/protein. The occurrence of S=1/2 EPR signals in the as isolated state and dithionite reduced state could be explained by the assumption that the 'prismane protein' had four redox states: the fully reduced [6Fe-6S] <sup>3+</SUP>state with the fingerprint prismane signal, an [6Fe-6S] <sup>4+</SUP>redox state with unknown spin state (S=0 or integer), the [6Fe-6S] <sup>5+</SUP>state with the molybdenum-like S=1/2 EPR signal and the fully oxidized [6Fe-6S] <sup>6+</SUP>redox state (S=0 or integer).The purification, chemical analysis and biochemical characterization of this 'prismane protein' are described in Chapter 4. The 'prismane' protein is a monomeric, cytoplasmic protein with a molecular mass of 52 kDa as estimated by sedimentation-equilibrium centrifugation. The protein contained 6.3±0.4 Fe and 6.2±0.7 S <sup>2-</SUP>per polypeptide. With polyclonal antibodies similar 'prismane' proteins were detected in <em>Desulfovibrio vulgaris</em> (Monticello) and <em>Desulfovibrio desulfuricans</em> (ATCC 27774). Using the N-terminal sequence and antibodies against this prismane protein Stokkermans and coworkers have sequenced the gene coding for this prismane protein as well as the homologous protein of <em>Desulfovibrio desulfuricans (ATCC</em> 27774).Further spectroscopic evidence for a new iron-sulfur cluster and strong support for the presence of a prismane core is presented in Chapter 5. The discovery that the [6Fe-6S] <sup>5+</SUP>redox state exhibited a spin mixture of approximately stoichiometric S=9/2 and substoichiometric molybdenum-like S=1/2 EPR signals confirmed the earlier hypothesis of four redox states of the prismane protein. Dye-mediated redox titrations and the observation of a g=16 signal with increased intensity in parallel-mode EPR for the [6Fe-6S] <sup>4+</SUP>redox state completed the following scheme:<img src="/wda/abstracts/i1679_1.gif" height="96" width="600"/>Multiple frequency EPR spectroscopy of the S=1/2 EPR signals showed that additional broadening indicative of nitrogen ligation was present. The line broadening caused by enrichment of the prismane protein with <sup>57</SUP>Fe was in agreement with ≈6 Fe per cluster. Quantitative high-resolution Mössbauer spectroscopy of the <sup>57</SUP>Fe enriched prismane protein revealed that both in the [6Fe-6S] <sup>3+</SUP>and the [6Fe-6S] <sup>5+</SUP>form the iron ions were inequivalent. <em>A</em> 4:2 ratio of quadrupole doublets was observed. The quadrupole splitting and isomer shift of the four irons ions were relatively invariant to the redox change of the cluster, while the two apparently more ionic irons had a more pronounced change from Fe <sup>2+</SUP>to Fe <sup>3+</SUP>character. Mössbauer spectroscopy at low temperatures and with applied magnetic fields indicated that the four and two iron ions were present in the same magnetically coupled structure. This led to a model in which the prismane structure is composed of a central set of four iron ions with a more ionic iron ion on each side. The more ionic iron ions could correlate with the nitrogen ligation as inferred from EPR studies.The unique EPR spectroscopic properties of the 'prismane protein' prompted investigation of dissimilatory sulfite reductase (desulfoviridin), a readily available iron-sulfur enzyme obtained during the isolation of Fe-hydrogenase from <em>Desulfovibrio vulgaris</em> (Hildenborough). The scrutiny for pure and electrophoretic homogeneous preparations of the desulfoviridin for EPR spectroscopic studies unexpectedly led to the discovery of a third, hitherto unrecognized 11 kDa subunit in this enzyme (Chapter 6). The γsubunit appeared to be tightly bound in the desulfoviridin complex for which a subunit composition of α _2β2γ2 was determined. N-terminal sequences and polyclonal antibodies against the α, βand γsubunits were obtained. The polyclonal antibodies allowed demonstration of the presence of homologous α, βand γsubunits in desulfoviridin-type dissimilatory sulfite reductases of three other <em>Desulfovibrio</em> species.Chapter 7 delineates the redox and spectroscopic results on the siroheme and S=9/2 EPR signals of desulfoviridin. By summation of the S=1/2 and S=5/2 EPR signals of the siroheme group it was shown that only 20% of the siroheme groups were metallated. The midpoint potential for the Fe <sup>2+</SUP>/Fe <sup>3+</SUP>transition of the main species of the siroheme was -295 mV. No significant amounts of EPR signals of normal iron-sulfur clusters were observed. Instead, several novel EPR signals with g= 17, g= 15. 1, g= 11.7 and g=9.0 were found in the as isolated oxidized form of the protein. These EPR signals were from a paramagnet with S=9/2. <em>A</em> stoichiometry of approximately 0.6 spin per αβγwas estimated. In a reductive redox titration the S=9/2 EPR signals disappeared with E _m =-205 mV. It was proposed that in the desulfoviridin -type dissimilatory sulfite reductase larger iron-sulfur clusters are present which give rise to the S=9/2 EPR signals. The demetallation of the siroheme and the S=9/2 EPR signals from an iron-sulfur cluster were in contradiction with the model of Siegel and coworkers for the sulfite reductase of <em>Escherichia coli,</em> in which coupling between a regular [4Fe-4S] <sup>2+</SUP>cubane and the Fe <sup>2+/3+</SUP>ion of the siroheme is proposed to explain spectroscopic properties.In Chapter 8 the redox and EPR spectroscopic properties of the nitrogenase MoFe protein from <em>Azotobacter vinelandii</em> are described. By controlled oxidation with dye-mediated redox titrations the long lasting controversy on the spin and redox states of the oxidized P-cluster iron-sulfur centers was solved. It turned out that oxidation of the P clusters could lead to two consecutive redox states, P <sup>OX1</SUP>and P <sup>OX2</SUP>. On oxidation of the dithionite reduced P-clusters by two electrons (E _m≈ -307 mV) first the P <sup>OX1</SUP>state is obtained with a weak g=12 EPR signal, which increased in intensity at higher temperature and sharpened and intensified>10 fold in parallel- mode EPR. This allowed assignment of the g=12 EPR signal to an excited state of a non-Kramers spin system (presumably S=3). Previous Mössbauer and MCD spectroscopic measurements appeared to have been made with this redox state. A second oxidation by one electron (E _m =+90 mV) led to the P <sup>OX2</SUP>redox state, which occurred as a spin mixture of S=1/2 and S=7/2 species. This redox state corresponded to the form obtained by the solid thionine oxidation procedure of Hagen and coworkers. Further oxidation of P <sup>OX2</SUP>redox state caused destruction of the iron-sulfur clusters and concommitant formation of S=9/2 and other high spin EPR signals.During the efforts to obtain highly-purified Fe-hydrogenase and prismane protein from <em>Desulfovibrio vulgaris</em> (Hildenborough) a black protein with an ultraviolet-visible spectrum reminiscent of rubredoxin-like iron-sulfur centers was obtained. Subsequent biochemical and EPR spectroscopic characterization (Chapter 9) indicated that this protein was similar but not identical to the protein rubrerythrin isolated by Moura and coworkers. The new protein was called nigerythrin due to its black color and hemerythrin-like EPR signal. Although rubrerythrin was originally reported to contain two rubredoxin-like and one dinuclear iron center, metal analyses and spin quantitation revealed that rubrerythrin and nigerythrin each contain two rubredoxin-like and two dinuclear iron centers per homodimer. The three redox transitions in both proteins had midpoint potentials higher than +200 mV. This suggested that both proteins have a non-redox role with all six iron ions in the ferrous state.In Chapter 10 a literature survey of non-integer and integer high spin systems in ironsulfur proteins is presented. In contrast to the well-documented occurrence of S=3/2 and S=2 spin states in [areas] <sup>1+</SUP>and [3Fe-4S] <sup>0</SUP>, respectively, the characterization of other, unusual iron-sulfur clusters with high spin states has not yet reached full maturity. The recent crystal structure of the MoFe protein of <em>Azotobacter vinelandii,</em> in which the FeMoco and P-clusters appeared to be larger clusters shows that the correlation between high spin states with structures other than the four basic iron-sulfur clusters indeed holds. The diversity of redox and spin states as observed for the prismane protein, desulfoviridin, carbonmonoxide dehydrogenase and Fe-hydrogenase indicates that besides the FeMoco and P-clusters other larger iron-sulfur clusters are present in biological systems.