Jacques Meyer

The coordination sphere of iron-sulfur clusters: lessons from site-directed mutagenesis experiments

Abstractu2002Cysteine is the ubiquitous ligand of iron-sulfur clusters in proteins, although chemical models have indicated that functional groups other than thiolates can coordinate iron in iron-sulfur compounds. Only a small number of naturally occurring examples of hydroxyl, histidinyl or carboxyl coordination have been clearly established but many others are suspected. Quite a few site-directed mutagenesis experiments have been aimed at replacing the cysteine ligands of iron-sulfur centers by other amino acids in various systems. The available data set shows that substituting one ligand, even by another functional residue, is very often destabilizing enough to impair cluster assembly; in some cases, the apoprotein cannot even be detected. One for one replacements have been demonstrated, but they have been so far almost exclusively confined to clusters with no more than one or two iron atoms. In contrast, changes of the cluster nuclearity or recruitment of free cysteine residues seem preferred ways for proteins containing larger clusters to cope with removal of a ligand, rather than using coordinating amino acids bearing different chemical functions. Furthermore, the possibility of replacing cysteines by other residues as ligands in iron-sulfur proteins does not uniquely depend on the ability of the cluster to accept other kinds of coordination than cysteinate; other factors such as the local flexibility of the polypeptide chain, the accessibility of the solvent and the electronic distribution on the active centers may also play a prominent role.

Comparison of carbon monoxide, nitric oxide, and nitrite as inhibitors of the nitrogenase from Clostridium pasteurianum

Abstract A comparative study of CO, NO, and nitrite as inhibitors of nitrogenase has been carried out. Confirming previous studies, we found that CO inhibits acetylene reduction, but not H 2 evolution nor ATP hydrolysis. On the other hand, NO and nitrite both inhibit acetylene reduction, H 2 evolution, and ATP hydrolysis. Nitrogenase inhibition by CO is readily reversible, whereas the effects of NO and nitrite are irreversible. NO was found to inactivate rapidly and irreversibly the Fe protein, but not the Mo-Fe protein. In the presence of NO, part of the iron of the Fe protein is complexed by bathophenanthrolinedisulfonate, which suggests that NO disrupts the Fe 4 S 4 cluster present in the protein. Like NO, nitrite reacts preferentially with the Fe protein, and it also induces complexation of the iron by bathophenanthrolinedisulfonate. We found that under the conditions normally used for the assay of nitrogenase, nitrite is reduced by dithionite. Even though the latter reaction proceeds at a very low rate, enough NO is evolved to inhibit nitrogenase. In view of the striking similarities between the inhibitory effects of NO and nitrite, we suggest that nitrogenase may be inhibited not by nitrite itself, but rather by the nitric oxide produced by the reduction of nitrite.

Refined crystal structure of the 2[4Fe-4S] ferredoxin from Clostridium acidurici at 1.84 Å resolution

The crystal structure of the 2[4Fe-4S] ferredoxin from Clostridium acidurici has been determined at a resolution of 1.84 A and refined to an R-factor of 0.169. Crystals belong to space group P4(3)2(1)2 with unit cell dimensions a = b = 34.44 A and c = 74.78 A. The structure was determined by molecular replacement using the previously published model of an homologous ferredoxin and refined by molecular dynamics techniques. The model contains the protein and 46 water molecules. Only two amino acid residues, Asp27 and Asp28, are poorly defined in the electron density maps. The molecule has an overall chain fold similar to that of other [4Fe-4S] bacterial ferredoxins of known structure. The two [4Fe-4S] clusters display similar bond distances and angles. In both of them the co-ordination of one iron atom (bound to Cys11 and Cys40) is slightly distorted as compared with that of the other iron atoms. A core of hydrophobic residues and a few water molecules contribute to the stability of the structure. The [4Fe-4S] clusters interact with the polypeptide chain through eight hydrogen bonds each, in addition to the covalent Fe-Scys bonds. The ferredoxin from Clostridium acidurici is the most typical clostridial ferredoxin crystallized so far and the biological implications of the newly determined structure are discussed.

High-yield chemical assembly of [2Fe-2X] (X = S, Se) clusters into spinach apoferredoxin: product characterization by resonance Raman spectroscopy

Abstract Conditions are described for the non-enzymic assembly of [2Fe-2S] or [2Fe-2Se] chromophores into spinach apoferredoxin with high yields (60% and 40%, respectively) under physiologically relevant conditions. The success of these reconstitution reactions was found to be critically dependent on the conditions used for the denaturation of native ferredoxin (0.5 M HCl, anaerobic conditions). Low-temperature resonance Raman spectra of native and Se-substituted spinach ferredoxin have been recorded and compared. Most spectral features are shifted to lower frequencies upon S ∗ → Se ∗ substitution, due to the larger atomic mass of selenium compared to sulfur. As a result, each of the spectra displays characteristic bands which are absent in the other. This observation has been used to show that some preparations of Se-substituted ferredoxin also contain [2Fe-2S] chromophores, most probably arising from residual inorganic sulfur bound to apoprotein prepared under non-optimal conditions. Quantitative estimations have shown that the presence of a few percent of residual sulfur in Se-substituted ferredoxin can be detected by resonance Raman spectroscopy. This technique has in addition been used to demonstrate that ferredoxin preparations containing both chalcogenides involve hybrid [2Fe-S-Se] clusters in addition to the [2Fe-2S] and [2Fe-2Se] ones.

Dynamics of Rhodobacter capsulatus [2Fe-2S] Ferredoxin VI and Aquifex aeolicus Ferredoxin 5 via Nuclear Resonance Vibrational Spectroscopy (NRVS) and Resonance Raman Spectroscopy

We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the Fe(2)S(2)(Cys)(4) sites in oxidized and reduced [2Fe-2S] ferredoxins from Rhodobacter capsulatus (Rc FdVI) and Aquifex aeolicus (Aa Fd5). In the oxidized forms, nearly identical NRVS patterns are observed, with strong bands from Fe-S stretching modes peaking around 335 cm(-1), and additional features observed as high as the B(2u) mode at approximately 421 cm(-1). Both forms of Rc FdVI have also been investigated by resonance Raman (RR) spectroscopy. There is good correspondence between NRVS and Raman frequencies, but because of different selection rules, intensities vary dramatically between the two kinds of spectra. For example, the B(3u) mode at approximately 288 cm(-1), attributed to an asymmetric combination of the two FeS(4) breathing modes, is often the strongest resonance Raman feature. In contrast, it is nearly invisible in the NRVS, as there is almost no Fe motion in such FeS(4) breathing. NRVS and RR analysis of isotope shifts with (36)S-substituted into bridging S(2-) ions in Rc FdVI allowed quantitation of S(2-) motion in different normal modes. We observed the symmetric Fe-Fe stretching mode at approximately 190 cm(-1) in both NRVS and RR spectra. At still lower energies, the NRVS presents a complex envelope of bending, torsion, and protein modes, with a maximum at 78 cm(-1). The (57)Fe partial vibrational densities of states (PVDOS) were interpreted by normal-mode analysis with optimization of Urey-Bradley force fields. Progressively more complex D(2h) Fe(2)S(2)S(4), C(2h) Fe(2)S(2)(SCC)(4), and C(1) Fe(2)S(2)(Cys)(4) models were optimized by comparison with the experimental spectra. After modification of the CHARMM22 all-atom force field by the addition of refined Fe-S force constants, a simulation employing the complete protein structure was used to reproduce the PVDOS, with better results in the low frequency protein mode region. This process was then repeated for analysis of data on the reduced FdVI. Finally, the degree of collectivity was used to quantitate the delocalization of the dynamic properties of the redox-active Fe site. The NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins.

Replacement Of Sulfur By Selenium In Iron—Sulfur Proteins

Publisher Summary This chapter describes the replacement of sulfur by selenium in iron–sulfur proteins. By its chemical properties, selenium is most similar to sulfur and occurs in the same valence states: −2, 0, +2, +4, and +6. However, the two elements display noteworthy differences relevant to the biochemistry of selenium. Selenium tends to be more stable than sulfur in its intermediate oxidation states and less stable in the extreme ones. Accordingly, selenate and selenite are relatively easy to reduce to the element, and selenides are more reactive (reducing) than sulfides. Selenium is most often found in biological systems in compounds such as selenols, diselenides, and selenoethers, which are usually more reactive than their sulfur counterparts, because of the greater polarity and lower strength of the C–Se, N–Se and O–Se bonds. Selenols are more acidic (usually ionized at neutral pH), are better nucleophiles, better leaving groups, and are more reducing than the corresponding thiols.

Structural differences between [2Fe-2S] clusters in spinach ferredoxin and in the “Red paramagnetic protein” from Clostridium pasteurianum. A resonance Raman study

The [2Fe-2S] ferredoxin (Red paramagnetic protein, RPP) from C. pasteurianum has been found to be composed of two identical subunits of 10,000 +/- 2 000 daltons, each containing a [2Fe-2S] cluster. Resonance Raman (RR) spectra of RPP have been obtained at 23 degrees K, and compared to those of spinach ferredoxin (Sp Fd). Ten modes of the [2Fe-2S] chromophore were observed in the 100-450 cm-1 range. Assignments of non fundamental modes in the 500-900 cm-1 range allowed correlations between fundamental stretching modes of RPP and Sp Fd. Although assuming a [2Fe-2S] structure, the chromophore of RPP differs from that of Sp Fd by its conformation and by a slight weakening of Fe-S bonds, involving both the inorganic core and the cysteine ligands.

Mössbauer, EPR, and MCD studies of the C9S and C42S variants of Clostridium pasteurianum rubredoxin and MCD studies of the wild-type protein

Abstract Rubredoxins contain a mononuclear iron tetrahedrally coordinated by four cysteinyl sulfurs. We have studied the wild-type protein from Clostridium pasteurianum and two mutated forms, C9S and C42S, in the oxidized and reduced states, with Mössbauer, integer-spin EPR, and magnetic circular dichroism (MCD) spectroscopies. The Mössbauer spectra of the ferric C42S and C9S mutant forms yielded zero-field splittings, D=1.2u2009cm−1, that are about 40% smaller than the D-value of the wild-type protein. The 57Fe hyperfine coupling constants were found to be ca. 8% larger than those of the wild-type proteins. The present study also revealed that the ferric wild-type protein has δ=0.24±0.01u2009mm/s at 4.2u2009K rather than δ=0.32u2009mm/s as reported in the literature. The Mössbauer spectra of both dithionite-reduced mutant proteins revealed the presence of two ferrous forms, A and B. These forms have isomer shifts δ=0.79u2009mm/s at 4.2u2009K, consistent with tetrahedral Fe2+(Cys)3(O-R) coordination. The zero-field splittings of the two forms differ substantially; we found D=−7±1u2009cm−1, E/D=0.09 for form A and D=+6.2±1.3u2009cm−1, E/D=0.15 for form B. Form A exhibits a well-defined integer-spin EPR signal; from studies at X- and Q-band we obtained gz=2.08±0.01, which is the first measured g-value for any ferrous rubredoxin. It is known from X-ray crystallographic studies that ferric C42S rubredoxin is coordinated by a serine oxygen. We achieved 75% reduction of C42S rubredoxin by irradiating an oxidized sample at 77u2009K with synchrotron X-rays; the radiolytic reduction produced exclusively form A, suggesting that this form represents a serine-bound Fe2+ site. Studies in different buffers in the pHu20096–9 range showed that the A:B ratios, but not the spectral parameters of A and B, are buffer dependent, but no systematic variation of the ratio of the two forms with pH was observed. The presence of glycerol (30–50% v/v) was found to favor the B form. Previous absorption and circular dichroism studies of reduced wild-type rubredoxin have suggested d-d bands at 7400, 6000, and 3700u2009cm−1. Our low-temperature MCD measurements place the two high-energy transitions at ca. 5900 and 6300u2009cm−1; a third d-d transition, if present, must occur with energy lower than 3300u2009cm−1. The mutant proteins have d-d transitions at slightly lower energy, namely 5730, 6100u2009cm−1 in form A and 5350, 6380u2009cm−1 in form B.

Replacement of sulfide by selenide in the [4Fe-4S] clusters of the ferredoxin fromClostridium pasteurianum

Abstract The sulfur atoms of the two [4Fe-4S] clusters present in the ferredoxin from C. pasteurianum have been replaced by selenium. The optical absorption spectrum of the Se-ferredoxin is slightly different from the spectrum of the native protein, but it displays the characteristic features of [4Fe-4X] ( X = S, Se) clustors. The reduced Se-ferredoxin can reduce hydrogenase, and the oxidized Se-ferredoxin can be reduced by hydrogenase in the presence of molecular hydrogen. This is the first report of sulfide substitution by selenide in an iron-sulfur protein containing [4Fe-4S] active sites.

Exceptional stability of a [2Fe-2S] ferredoxin from hyperthermophilic bacterium Aquifex aeolicus.

Aquifex aeolicus is the only hyperthermophile that is known to contain a plant- and mammalian-type [2Fe-2S] ferredoxin (Aae Fd1). This unique protein contains two cysteines, in addition to the four that act as ligands of the [2Fe-2S] cluster, which form a disulfide bridge. We have investigated the stability of Aae Fd1 with (wild-type) and without (C87A variant) the disulfide bond, with respect to pH, thermal and chemical perturbation, and compared the results to those for the mesophilic [2Fe-2S] ferredoxin from spinach. Unfolding reactions of all three proteins are irreversible due to cluster decomposition in the unfolded state. Wild-type and C87A Aae Fd1 proteins are extremely stable: unfolding at 20 degrees C requires high concentrations of the chemical denaturant and long incubation times. Moreover, their thermal-unfolding midpoints are 40-50 degrees higher than that for spinach ferredoxin (pH 7). The stability of the Aae Fd1 protein is significantly lower at pH 2.5 than pH 7 and 10, suggesting that ionic interactions play a role in structural integrity. Interestingly, the iron-sulfur cluster in C87A Aae Fd1 rearranges into a transient species with absorption bands at 520 and 610 nm, presumably a linear three-iron cluster, in the high-pH unfolded state.