Incorporation of either molybdenum or tungsten into formate dehydrogenase from Desulfovibrio alaskensis NCIMB 13491; EPR assignment of the proximal iron-sulfur cluster to the pterin cofactor in formate dehydrogenases from sulfate-reducing bacteria

Abstract

We report the characterization of the molecular properties and EPR studies of a new formate dehydrogenase (FDH) from the sulfate-reducing organism Desulfovibrio alaskensis NCIMB 13491. FDHs are enzymes that catalyze the two-electron oxidation of formate to carbon dioxide in several aerobic and anaerobic organisms. D. alaskensis FDH is a heterodimeric protein with a molecular weight of 126±2 kDa composed of two subunits, a=93±3 kDa and b=32±2 kDa, which contains 6±1 Fe/molecule, 0.4±0.1 Mo/molecule, 0.3±0.1 W/molecule, and 1.3±0.1 guanine monophosphate nucleotides. The UV-vis absorption spectrum of D. alaskensis FDH is typical of an iron-sulfur protein with a broad band around 400 nm. Variable-temperature EPR studies performed on reduced samples of D. alaskensis FDH showed the presence of signals associated with the different paramagnetic centers of D. alaskensis FDH. Three rhombic signals having g-values and relaxation behavior characteristic of [4Fe-4S] clusters were observed in the 5-40 K temperature range. Two EPR signals with all the g-values less than two, which accounted for less than 0.1 spin/protein, typical of mononuclear Mo(V) and W(V), respectively, were observed. The signal associated with the W(V) ion has a larger deviation from the free electron g-value, as expected for tungsten in a d 1 configuration, albeit with an unusual relaxation behavior. The EPR parameters of the Mo(V) signal are within the range of values typically found for the slow-type signal observed in several Mo-containing proteins belonging to the xanthine oxidase family of enzymes. Mo(V) resonances are split at temperatures below 50 K by magnetic coupling with one of the Fe/S clusters. The analysis of the inter-center magnetic interaction allowed us to assign the EPR-distinguishable iron-sulfur clusters with those seen in the crystal structure of a homologous enzyme.

Structural and Electron Paramagnetic Resonance (EPR) Studies of Mononuclear Molybdenum Enzymes from Sulfate-Reducing Bacteria

“…Examples of temperaturedependent magnetic interactions have been observed in all aldehyde oxidoreductases from SRB and in Da Fdh, in which the Mo V ion of the active site is coupled to the closest FeS center. 20,37 One of the strategies to correlate EPR with crystal data can be used when the redox potential of the interacting centers with Mo V ions are different, even when this difference is as small as 50 mV, as is the case for Da Aor. 13 Da Aor (and all aldehyde oxidoreductases from SRB) shows two EPR signals named FeS I (g 1 ) 1.916, g 2 ) 1.934, and g 3 ) 2.021) and FeS II (g 1 ) 1.900, g 2 ) 1.970, and g 3 ) 2.066), which are associated with the two [2Fe-2S] clusters detected from X-ray data in aldehyde reductases (Figure 3).…”

“…38 The results of this simulation as a function of the relaxation time T 1 of the faster relaxing center is shown in Figure 8B. Details of the model employed in the simulation are given in ref 20, and the dependence with temperature of The double-headed arrows connect microstates separated by one-electron reduction/oxidation (P 0 and P 11 represent the fully oxidized and reduced states, respectively) (b) EPR spectrum of Da Aor samples at -450 and -334 mV. Simulation sim-a was obtained considering that FeS I is the center closer to Mo, whereas sim-b was obtained considering that FeS II is the closer one.…”

Mo and W bis-MGD enzymes: nitrate reductases and formate dehydrogenases

“…D. desulfuricans Fdh contains Mo at the active site [21,22], but D. alaskensis Fdh can incorporate either Mo or W [23]. The Fdh from SRB present, in addition to the large subunit, a second subunit ($29 kDa), which was shown by X-ray studies of the D. gigas enzyme to contain four [4Fe-4S] clusters [19,20].…”

“…Fdh from the SRB D. desulfuricans and D. alaskensis have been purified and characterized, and are closely related to D. gigas W-Fdh [23]. D. desulfuricans Fdh contains Mo at the active site [21,22], but D. alaskensis Fdh can incorporate either Mo or W [23].…”

“…A second broader EPR signal (Fe/S II) appears below 30 K for all the members (mean g-values: g 1 =2.067, g 2 =1.921, g 3 =1.873), and a third broad signal can be discernible in D. alaskensis Fdh below 20 K. D. alaskensis Fdh presents the Mo site magnetically coupled to one of the iron-sulfur clusters, a fact that was never seen before in proteins having a bis-pterin cofactor with a guanine dinucleotide (bis-MGD) coordination. The analysis of the inter-center magnetic interaction showed that Fe/S I is the center closest to the active site in Fdh from SRB [23].…”