Quantitative EPR of an S= 7/2 system in thionine‐oxidized MoFe proteins of nitrogenase

Abstract

Thionine-oxidized nitrogenase MoFe proteins from Azotohucter vinelundii. Azotobacter cliroococcurn and Klehsiella pneurnoniae exhibit excited-state EPR signals with g = 10.4, 5.8 and 5.5 with a maximal amplitude in the temperature range of 20-50 K. The magnitude of these effective g values, combined with the temperature dependence of the peak area at g = 10.4 from 12 K to 86 K, are consistent with an S = 7/2 system with spin01 cm-' and g = 2.00. This interpretation predicts nine additional effective g values some of which have been detected as broad features of low intensity at g z 10, z 2.5 and z 1.8. The S = 7/2 EPR is ascribed to the multi-iron exchange-coupled entities known as the P clusters.Quantification relative to the S = 312 EPR signal from dithionite-reduced MoFe protein indicates a stoichiometry of one P cluster per FeMo cofactor. Two possible interpretations for these observations, together with data from the literature, are proposed. In the first model there are two P clusters per tetrameric MoFe protein. Each P cluster encompasses approximately 8Fe ions and releases a total of three electrons on oxidation with excess thionine. In the second model the conventional view of four P clusters, each containing approximately 4Fe, is retained. This alternative requires that following one-electron oxidation, the P clusters factorize into two populations, P, and Pb, only one of which is further oxidized with thionine resulting in the S = 7 / 2 system. Both models require eight-electron oxidation of tetrameric MoFe protein to reach the S = 7/2 state.

Redox properties and EPR spectroscopy of the P clusters of Azotobacter vinelandii MoFe protein

“…Solid-thionine-oxidation was according to Hagen et al [23] : 150 pl dithionite-free MoFe protein solution (97 mg/ ml) was anaerobically incubated with 1.5 mg thionine (Kodak Eastman). Samples oxidized with stoichiometric amounts of dissolved thionine (cf.…”

“…These clusters would further differ from the classical cubanes in that, upon oxidation with thionine, they have an 'EPR-silent' uniaxial doublet ground state within a S = 3/2, 5/2, 712 or 9/2 multiplet [9, 121. Support for this model was found by low-temperature magnetic circular dichroism (MCD) studies, from which it was concluded that the ground state was an electronic doublet with an estimated In the alternative model of Hagen and coworkers [23] the 4 X 4-Fe P cluster concept was redefined after the discovery of S = 7/2 EPR signals in solid-thionine-oxidized MoFe proteins. On the basis of an observed stoichiometry of approximately two S = 7/2 spidprotein, it was proposed that a P cluster may be a larger grouping of some eight Fe atoms; this implies a MoFe protein with only two P clustershetramer.…”

“…1C). Liquid nitrogen boil-off temperatures were found to give the most reproducible readings for the g = 10.4 amplitude, although the optimal temperature for the g = 10.4 EPR signal of the I 2 1/2> doublet is some 40 K [23]. At 94 K the detection limit for the g = 10.4 S = 7/2 EPR signal corresponded to a MoFe protein concentration of 1 mg/ml(5 pM).…”

“…Hagen and coworkers showed that for D = -3.7 cm-' the amplitude of the g = 10.4 EPR signal from the 12 1/2> doublet perfectly obeyed a S = 7/2 Boltzmann distribution function in the 4.2-100 K range [23]. Although not stated in [23], the temperature dependence of the g = 5.8 EPR signal from the I t 3/2 > doublet can be used to obtain a similar D value with a normal S = 7/2 Boltzmann distribution function. The temperature dependence of two doublets thus indicates that the S = 7/2 multiplet is energetically isolated from both lower and higher electronic spin states.…”

“…Equilibrium redox titration of the S = 7/2 EPR signal Hagen and coworkers have shown that A. vinelandii MoFe protein exhibits S = 712 EPR signals after treatment with solid thionine [23]. To demonstrate whether solid thionine induces these signals by binding or by oxidation, we performed a mediated equilibrium redox titration of the S = 7/2 EPR signals of A. vinelandii MoFe protein (Fig.…”