The active centers of adenylylsulfate reductase from Desulfovibrio gigas. Characterization and spectroscopic studies

Abstract

In order to utilize sulfate as the terminal electron acceptor, sulfate-reducing bacteria are equipped with a complex enzymatic system in which adenylylsulfate (AdoPSO4) reductase plays one of the major roles, reducing AdoPSO4 (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 (approximately 400 kDa). It is an iron-sulfur, flavin-containing 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 57Fe-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, H2/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 57Fe-enriched D. gigas AdoPSO4 reductase demonstrated unambiguously the presence of two 4Fe clusters. Center II has a redox potential less than or equal to 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.