Circular dichroism and magnetic circular dichroism of nitrogenase proteins

Abstract

Circular dichroism (CD) The enzyme nitrogenase (N2ase) has been isolated and purified from various nitrogen-fixing organisms and is currently the subject of intensive investigations (1-4). Active N~ase systems have been shown to consist of two essential metalloproteins-the MoFe protein and ;(20)(21)(22)(23)(24)(25)(26)(27)(28) and the Fe protein (containing -4 Fe and t4 S2-)-which together, in the presence of a suitable electron donor, catalyze ATP-dependent reduction of N2 to NH3. Existing evidence suggests that electrons derived from the primary reductant are transferred via the Fe protein to the MoFe protein, which is believed to provide the site for N2 binding (1-5).Despite study by various spectroscopic and other techniques, important aspects of the structure and catalytic role of the metal centers in N2ase remain to be elucidated (1-4). Recent systematic studies of the circular dichroism (CD) and magnetic circular dichroism (MCD) of simple iron-sulfur proteins (6, 7) have shown that CD and MCD can be useful in characterizingThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 2585 iron-sulfur cluster type, oxidation level, and protein environment and that more information is afforded by these probes than by unpolarized absorption spectroscopy. Because the Fe and MoFe proteins evidently contain iron-sulfur clusters-albeit unconventional in many respects-we have undertaken to explore the value of CD and MCD in the study of N2ase. Electronic spectroscopy has so far found relatively limited application in the study of this enzyme (1-4). Electronic absorption spectra of the two components are almost featureless.CD spectra have been obtained in the polypeptide.absorption region (wavelengths <300 nm) (8, 9), but CD was reported to be absent at longer wavelengths in both N2ase components from Azotobacter chroococcum (10) and was also undetectable in Fe protein from Klebsiella pneumoniae (8). Very weak visible CD has been reported in the Klebsiella MoFe protein; however, the spectrum was not given (8). No MCD work has been published.We report here spectra demonstrating that CD and MCD are observable in both N2ase components across the near-infrared-visible-near-ultraviolet spectral region

Resonance Raman studies of the [4Fe‐4S] to [2Fe‐2S] cluster conversion in the iron protein of nitrogenase

“…In relation to the first point, it offers an alternative interpretation for the dramatic change in the CD spectrum that is observed on MgATP binding to oxidized nitrogenase Fe-proteins [9]. Proteins containing oxidized [2Fe-2S12 + clusters exhibit much more intense CD spectra than those containing [4Fe-4S12+ clusters [20].…”

Docking of nitrogenase iron‐and molybdenum‐iron proteins for electron transfer and MgATP hydrolysis: The role of arginine 140 and lysine 143 of the Azotobacter vinelandii iron protein

“…This state would be analogous to the heterologous Clostridium pasteurianurn FeP-A. vinelandii MoFeP complex that catalyzes MgATP hydrolysis, but not electron transfer (Smith et al, 1976;Emerich & Burris, 1978 (Orme-Johnson et al, 1972;Zumft et al, 1972), a change in the midpoint potential of the cluster by -150 mV (Watt et al, 1986), changes in the CD spectrum (Stephens et al, 1979), shifts in the NMR resonances of protons magnetically coupled to the paramagnetic [4Fe-4S] cluster (Meyer et al, 1988), changes in scattering of X-rays (Chen et al, 1994), and in Fez+ chelation (Walker & Mortenson, 1974). This conformation of the FeP would be required for the formation of the second, MgATP hydrolytic state, with the MoFeP.…”

Sequence of the nifD gene coding for the α subunit of dinitrogenase from the cyanobacterium Anabaena

“…If all four Fe4S4 centers are ligated totally by cysteine, they would require a total of 16, leaving 4 cysteines for bonding to the pair of MoFe centers or to the poorly characterized S center, which probably contains two Fe atoms, or to both. Neither the Fe4S4 centers nor the S center corresponds to known, typical centers in situ (26). Thus, it is possible that there are some non-thiolate bonds to the Fe-S centers, in which case a larger number of cysteines would be available for liganding to the MoFe cofactors.…”