Abstract
The structural and electronic properties of the [2Fe-2S] clusters in reduced putidaredoxin, Spinacea oleracea ferredoxin, and Clostridium pasteurianum [2Fe-2S] ferredoxin have been investigated by resonance Raman and variable temperature magnetic circular dichroism spectroscopies. Both techniques are shown to provide diagnostic fingerprints for identifying [2Fe-2S]+ clusters in more complex multicomponent metalloenzymes. The Fe-S stretching modes of oxidized and reduced putidaredoxin are assigned via 34S and D2O isotope shifts and previous normal mode calculations for adrenodoxin (Han, S., Czernuszewicz, R. S., Kimura, T., Adams, M. W. W., and Spiro, T. G. (1989) J. Am. Chem. Soc. 111, 3505-3511). The close similarity in the resonance Raman spectra of reduced [2Fe-2S] centers, in terms of both the vibrational frequencies and enhancement profiles of the Fe-S stretching modes, permits these assignments to be generalized to all clusters of this type. Modes primarily involving Fe(III)-S(Cys) stretching are identified in all three reduced [2Fe-2S] proteins, and the frequencies are rationalized in terms of the conformation of the cysteine residues ligating the Fe(III) site of the localized valence reduced cluster. D2O isotope shifts indicate few, if any, amide NH-S hydrogen bond interactions involving the cysteines ligating the Fe(III) site. Preliminary resonance Raman excitation profiles suggest assignments for the complex pattern of electronic bands that comprise the low temperature magnetic circular dichroism spectra of the reduced proteins. S----Fe(III) and Fe(II)----S charge transfer, Fe d-d, and Fe(II)----Fe(III) intervalence bands are identified.
Highlights
The structural andelectronic properties of the [2Fe251 clusters in reduced putidaredoxin, Spinacea oleracea ferredoxin, and Clostridium pasteurianum [2Fe251 ferredoxin have been investigated by resonance Raman andvariabletemperature magnetic circular dichroism spectroscopies
Resonance Raman has emerged as a sensitive structural probe for oxidized[2Fe-2SI2+clusters in proteins, and reliable vibrational assignments based on extensive IR and Raman studies of isotopically labeled synthetic analog complexes are available (11,12).Based on the arrangement of conserved cysteines, three distinct classes have been proposed for [2Fe2S] proteins with complete cysteinyl coordination (13):planttype Fds, hydroxylase-type proteins, and bacterial [2Fe-2S] Fds
The observed differences in the Fe-S stretching frequencies amongthese proteinshave been rationalized in termsof minor variations in the Fe-S bond strengths and differences in the Fe-St-C-C dihedral angles which govern the extent of mixing between cysteinyl St-C-Cbending modes and Fe-St stretching modes (St indicates terminalor cysteinyl S) (11, 12)
Summary
S. okracea (spinach) Fd was obtained from Sigma and further by direct analogy to adrenodoxin (12). Bandintensities in purified according to Petering and Palmer (15) to give protein with resonance Raman spectra depend critically on the symmetry an absorption ratio A420/Am = 0.40. Shifts indicate close structural similarity between the [2Fe2S] clustersin putidaredoxin and adrenodoxin. This is in accord with the high degree of sequence homology (13), and the marked similarities inboth midpoint potentials Raman spectra were recorded with an Instruments SA UlOOO spectrometer fitted with a cooled RCA 31034photomultiplier tube, using lines from a Coherent Innova 100 10-watt argon ion laser or Coherent. Fe-S stretchingfrequencies, 34Sabnd DzO isotope shifts, and Vibrational assignments for the [2Fe-2SI2+clusters in oxidized adrenodoxin, putidaredoxin, C. pasteurianum (Cp) [2Fe-2S] Fd,and. Band positions were calibrated using the excitation frequency, and the principal bands ofCC14 and are accurate to +. 1 cm". 34Sband
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