Iron-sulfur proteins spectrum

Fig. 6. Electron paramagnetic resonance signal showing the g= 1.94 characteristic of the dithionite-reduced spinach ferredoxin, a plant-type iron-sulfur protein. Spectrum taken at 20 °K...

A preliminaiy characterization of a new iron—sulfur protein isolated from Desulfovibrio vulgaris Hildenborough was reported in 1989 124). The protein contained approximately 6 iron and 6 inorganic sulfur atoms per molecule. The FPR spectrum of the dithionite reduced protein exhibited an S = signal similar to what was found for synthetic compounds with a [6Fe-6S] core (prismane core). No other FPR signals were reported at this time, and based on the observed similarity it was suggested that this peculiar iron-sulfur protein contained a [6Fe-6S] cluster. Because it had no known function, the pro-... 

In 1971, adrenodoxin, an iron-sulfur protein with a single tyrosine residue and no tryptophan was shown to fluoresce at 331 nm upon 280-nm excitation at neutral pH/20 1 On cooling from room temperature to 77 K, the emission maximum shifts to 315 nm. The redox state of the iron does not have any effect on the tyrosine emission. From these results, an exciplex between the excited singlet state of tyrosine and an unidentified group was suggested as the cause of the anomalous emission energy/2031 Later studies have shown that the excitation spectrum is a red-shifted tyrosine spectrum, that removal of the iron to form the apoprotein has no effect on the emission, and that heat, low pH, guanidine hydrochloride, urea, and LiCl all cause the emission... 

Both stopped-flow and rapid freeze quench kinetic techniques show that the substrate reduces the flavin to its hydroquinone form at a rate faster than catalytic turnover Reoxidation of the flavin hydroquinone by the oxidized Fe4/S4 center leads to formation of a unique spin-coupled species at a rate which appears to be rate limiting in catalysis. Formation of this requires the substrate since dithionite reduction leads to flavin hydroquinone formation and a rhombic ESR spectrum typical of a reduced iron-sulfur protein . The appearance of such a spin-coupled flavin-iron sulfur species suggests the close proximity of the two redox centers and provides a valuable system for the study of flavin-iron sulfur interactions. The publication of further studies of this interesting system is looked forward to with great anticipation. 

Indeed it may be that 3Fe clusters are always extruded as [2Fe-2S] clusters, an observation that would have structural implications. The use of resonance Raman and MCD spectroscopy814 has allowed valuable distinctions to be made between these clusters, while ESR techniques seem to be of particular value. Thus, the first indication of a 3Fe cluster in an iron-sulfur protein has usually been the observation of a g = 2.01 signal in the ESR spectrum, but it should be noted that there may be confusion with oxidized HiPIP clusters. 

Interesting results have been obtained for mitochondrial particles prepared from the yeast Candida utilis grown in a medium containing 57Fe. This allowed hyperfine interactions for 57Fe atoms in the ESR spectrum to be characterized. Certain of the iron-sulfur proteins are also proton pumps. 

These spectra, taken at variable temperatures and a small polarizing applied magnetic field, show a temperature-dependent transition for spinach ferredoxin. As the temperature is lowered, the effects of an internal magnetic field on the Mossbauer spectra become more distinct until they result at around 30 °K, in a spectrum which is characteristic of the low temperature data of the plant-type ferredoxins (Fig. 11). We attribute this transition in the spectra to spin-lattice relaxation effects. This conclusion is preferred over a spin-spin mechanism as the transition was identical for both the lyophilized and 10 mM aqueous solution samples. Thus, the variable temperature data for reduced spinach ferredoxin indicate that the electron-spin relaxation time is around 10-7 seconds at 50 °K. The temperature at which this transition in the Mossbauer spectra is half-complete is estimated to be the following spinach ferredoxin, 50 K parsley ferredoxin, 60 °K adrenodoxin, putidaredoxin, Clostridium. and Axotobacter iron-sulfur proteins, 100 °K. 

Fia. 8. Absorption spectrum of the soluble iron-sulfur protein (4 mg/ml) isolated from complex I. Dashed line, after treatment with dithionite dotted line, after treatment with sodium mersalyl to destroy the iron-sulfur chromophore. From Hatefi et al. (Si),... 

The resolved complex is composed of two fractions, a soluble part, which comprises about 15% of complex I proteins, and a water-insoluble part consisting of the rest of the protein and the bulk of complex I lipids. The soluble fraction is easily separated from the insoluble material by centrifugation. Upon fractionation with ammonium sulfate, it yields a soluble flavoprotein containing iron and labile sulfide and a dark brown protein, which contains large amounts of iron and labile sulfide but no flavin. The latter appears to be an iron-sulfur protein and exhibits an EPR signal which is characteristic of iron-sulfur center 2 of intact complex I (46). Its absorption spectrum is shown in Fig. 8. The insoluble fraction also contains equimolar amounts of iron and labile sulfide and little or no flavin. 

The [4Fe-4S] cluster in AOR and FOR is paramagnetic in its reduced form and displays characteristic EPR resonances, although the spin relaxation rate is very fast and the spectra are observed only at very low temperatures (see Iron-Sulfur Proteins). Hence, at 4K, the EPR spectrum of AOR is dominated by resonances from its reduced Fe-S cluster. However, this gives rise to a rhombic signal from a S = 3/2 ground state, while reduced 4Fe-clusters typically have an S = 1/2 ground state. In fact, while mixed spin, S = 3/2 and S = 1/2 reduced [4Fe-4S] clusters are sometimes observed in some iron-sulfur proteins (see Iron-Sulfur Proteins), clusters that have exclusively S = 3 /2 ground states are so far unique to AOR. Indeed, the reduced 4Fe-cluster of FOR has a mixed spin state with S = 3/2 (80%) and S = 1/2 (20%) components. The factors that determine the spin state of a [4Fe-4S] center have yet to be elucidated. 

Detailed studies on and Fb have been hampered by the property that their EPR spectra are not additive. This property has been attributed to a magnetic interaction between reduced F and Fg, indicating that they are very close to each other. The values of F and Fg are -540 and -590 mV respectively in spinach PS I particles. Their values are always in that range, but their relative values vary in different plant species for example, F has a more negative than Fb in barley and in a halophilic alga. The shape and temperature dependence of the EPR spectra of F and Fb are typical of iron-sulfur proteins. They are considered to be 4Fe-4S centers, since after modification by dimethyl sulfoxide their spectrum is characteristic of 4Fe-4S centres and because their Mossbauer spectra are also in agreement with that attribution. The presence of 10-12 Fe-S pairs in each PS I centre is compatible with this assignment (for reviews, see Refs. 25 and 26). 

At room temperature, flash absorption studies revealed that an electron acceptor designated Aj was functioning under conditions where F and Fg were presumably reduced [37]. The state (P-700, A2 ) is formed upon flash excitation and recombines with tiu — 250 jUS. The difference spectrum due to its formation was analysed into contributions of P-700 and A2. The latter includes mainly a small and broad bleaching around 430 nm, and perhaps some absorption shifts in the red. These absorption properties, together with the disappearance of the A2 absorption signal when iron-sulfur proteins are denatured [38,39], indicate that Aj may be an iron-sulfur centre. 

A third species, Fx, the spectrum of which considerably deviates from that of a ferredoxin, is observed under highly reducing conditions [43,44], From Mossbauer studies it was calculated that Fx is a 4Fe-4S iron-sulfur protein [45], It is still not quite certain, however, whether under physiological conditions Fx really acts as an obligatory electron acceptor. In spite of the above-mentioned uncertainties, EPR is the only technique that is capable of furnishing detailed information on the various iron-sulfur protein acceptors their optical absorbance difference spectra all show a rather uninformative weak band around 430 nm,... 

The membrane-bound iron-sulfiir centers were discovered by Dick Malkin and Alan Bearden in 1971 in spinach chloroplasts using EPR spectroscopy. Since the EPR spectrum was found to resemble that of the iron-sulfur protein ferredoxin and since the soluble ferredoxin had already been removed from the chloroplast sample used in the measurement, the substance represented by the newly found EPR spectrum was initially called membrane-bound ferredoxin. And since the iron-sulfur center was also found to be photo-reducible at cryogenic temperature, it was therefore suggested that it was the primary electron acceptor of photosystem I. 

In an attempt to characterize the membrane-bound iron-sulfur proteins, Malkin, Aparicio and Amon isolated the iron-sulfur protein by acetone extraction of chloroplasts which had previously been freed of soluble ferredoxin. They estimated the molecular mass of the protein to be 8 kDa. They also found that its absorption spectrum displayed no characteristic features. The EPR spectrum of the isolated protein was, however, quite different from that of the protein in the native, membrane-bound state. 

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