The active centers of adenylylsulfate reductase from Desulfovibrio gigas

In order to utilize sulfate as the terminal electron acceptor, sulfate‐reducing bacteria are equipped with a complex enzymatic system in which adenylylsulfate (Ado P SO 4 ) reductase plays one of the major roles, reducing Ado P SO 4 (the activated form of sulfate) to sulfite, with release of AMP. The enzyme has been purified to homogeneity from the anaerobic sulfate reducer Desulfovibrio gigas . The protein is composed of two non‐identical subunits (70 kDa and 23 kDa) and is isolated in a multimeric form (∼ 400 kDa). It is an iron‐sulfur, flavincontaining protein, with one FAD moiety, eight iron atoms and a minimum molecular mass of 93 kDa. Low‐temperature EPR studies were performed to characterize its redox centers. In the native state, the enzyme showed an almost isotropic signal centered at g = 2.02 and only detectable below 20 K. This signal represented a minor species (0.10–0.25 spins/mol) and showed line broadening in the enzyme isolated from 57 Fe‐grown cells. Addition of sulfite had a minor effect on the EPR spectrum, but caused a major decrease in the visible region of the optical spectrum (around 392 nm). Further addition of AMP induced only a minor change in the visible spectrum whereas major changes were seen in the EPR spectrum; the appearance of a rhombic signal at g values 2.096, 1.940 and 1.890 (reduced Fe‐S center I) observable below 30 K and a concomitant decrease in intensity of the g = 2.02 signal were detected. Effects of chemical reductants (ascorbate, H 2 /hydrogenase‐reduced methyl viologen and dithionite) were also studied. A short time reduction with dithionite (15 s) or reduction with methyl viologen gave rise to the full reduction of center I (with slightly modified g values at 2.079, 1.939 and 1.897), and the complete disappearance of the g = 2.02 signal. Further reduction with dithionite produces a very complex EPR spectrum of a spin–spin‐coupled nature (observable below 20 K), indicating the presence of at least two iron‐sulfur centers, (centers I and II). Mössbauer studies on 57 Fe‐enriched D. gigas Ado P SO 4 reductase demonstrated unambiguously the presence of two 4Fe clusters. Center II has a redox potential 400 mV and exhibits spectroscopic properties that are characteristic of a ferredoxin‐type [4Fe‐4S] cluster. Center I exhibits spectra with atypical Mössbauer parameters in its reduced state and has a midpoint potential around 0 mV, which is distinct from that of a ferredoxin‐type [4Fe‐4S] cluster, suggesting a different structure and/or a distinct cluster‐ligand environment.