Resonance Raman spectra of spinach ferredoxin and adrenodoxin and of analog complexes

Abstract

Resonance Raman spectra of oxidized spinach ferredoxin and adrenodoxin show six and seven bands in the Fe-S stretching region (280-430 cm"1). Reconstitution of the proteins with labile 34S using rhodanese and 34SS032" produced isotope shifts allowing identification of all four Fe2S2 bridging modes and two (three for adrenodoxin) of the four expected Fe-S(Cys) terminal modes. Similar spectra are observed for the analogue complexes Fe2S2(S2-o-xyl)22~(S2-o-xyl = o-xylylenedithiolate) and Fe2S2Cl42", but they show the expected Dih selection rules, and IR-active modes are absent or weak in the Raman spectra.All eight (bridging plus terminal) stretching modes were located via IR as well as Raman spectroscopy and were calculated with reasonable accuracy by using a Urey-Bradley force field scaled to the crystallographically determined interatomic distances. The spinach ferredoxin and Fe2S2(S2-oxyl)22~frequencies are very similar, but adrenodoxin shows appreciably different terminal frequencies, suggestive of conformational differences of the ligated cysteines. The strong activation of IR modes in the protein Raman spectra implies a protein-induced inequivalence of the two ends of the Fe2S2 complex, which may contribute to the known localization of the added electron in the reduced form. This influence is suggested to be due to enhanced H bonding to the cysteine sulfur atoms at one end of the complex, consistent with available crystal structure data. Reduction of adrenodoxin shifts the bridging frequencies 16-24 cm'1 to lower frequency, consistent with the expected weakening of the bridging bonds. The terminal modes, however, were unobserved, plausibly due to a loss of enhancement associated with weakened terminal S -* Fe charge transfer intensity, except for a weak band at 308 cm"1. At higher frequencies, 550-850 cm'1, the protein spectra showed weak bands associated with the Fe-S overtone and combination levels. Previously reported spin-ladder Raman bands of adrenodoxin and spinach ferredoxin were not observed and are attributed to artifacts produced by laser-induced protein damage. A very broad (~200 cm'1) and weak feature at ~1000 cm"1 in the adrenodoxin spectrum might possibly be due to the S = 0 -1> 1 electronic transition of the spin-coupled Fe3+ ions.

Raman and infrared spectroscopic evidence for the structural changes of the 2Fe 2S cluster and its environment during the interaction of adrenodoxin and adrenodoxin reductase

“…Raman spectra of Adx [20], [21], [24] The Raman spectra obtained for the Adx WT correlate with the previous Raman spectroscopic studies and the band assignments made by analogy to these studies, are summarized in Table 1. [21], [20], [25] The WT data was directly compared to the Adx (4-108) C-terminus variant, a mutant where the first 3 amino acids and the amino acids after position 108 have been deleted.…”

“…[21], [20], [25] The WT data was directly compared to the Adx (4-108) C-terminus variant, a mutant where the first 3 amino acids and the amino acids after position 108 have been deleted. In the spectra of this so-called truncated form of Adx ( Figure 2B), the bridging and terminal vibrational modes, seen at 412 cm -1 and 323 cm -1 , shift by 3-5 cm -1 to lower wavenumbers, indicating that the absence of the C terminus slightly changes the geometry of the [2Fe-2S] cluster.…”

“…It is also noted that a small signal at 373 cm -1 was attributed to a Fe-S b mode with B 2g symmetry in data of Adx excited at 458 nm. [21] Alternatively, the terminal and bridging (Fe-S t and Fe-S b ) stretching vibrations in the complex may be affected by a change in hydrogen bonding. Indeed, the presence of strong hydrogen bonding interactions was previously described between residues bridging the [2Fe-2S] cluster of Adx and the isoalloxazine ring of the FAD of AdR in the crystal structure of the Adx WT/AdR complex.…”

“…The spectra correspond to previously obtained data. [20,21] Han et al examined the Raman excitation profiles and the wavelength selectivity of ferrodoxin reporting that the terminal modes are well excited in the green-yellow region, whereas the bridging modes would be well enhanced in the blue. [20] The resonance of the CT bands at 514 nm was thus possible.…”

Adventures in Bioinorganic Chemistry

“…Roman Czernuszewicz and Mike Johnson led this effort. [31][32][33][34] We studied cobalamins, as models for vitamin-B 12 -containing enzymes. 35,36 In the early work, photolysis from the Raman laser precluded studies of the physiologically relevant alkyl cobalamins, but later we discovered how to circumvent this problem using frozen solutions, leading to mechanistic studies of important enzymes.…”