The periplasmic Fe-hydrogenase from Desulfovibrio vulguris (Hildenborough) contains three ironsulfur prosthetic groups : two putative electron transferring [4Fe-4S] ferredoxin-like cubanes (two Fclusters), and one putative Fe/S supercluster redox catalyst (one H-cluster). Combined elemental analysis by proton-induced X-ray emission, inductively coupled plasma mass spectrometry, instrumental neutron activation analysis, atomic absorption spectroscopy and colorimetry establishes that elements with Z > 21 (except for 12-15 Fe) are present in 0.001-0.1 mol/mol quantities, not correlating with activity. Isoelectric focussing revmIs the existence of multiple charge conformers with PI in the range 5 7-6.4. Repeated re-chromatography results in small amounts of enzyme of very high H,-production activity determined under standardized conditions (z 7000 Ujmg). The enzyme exists in two different catalytic forms: as isdated the protein is 'resting' and 02-insensitive; upon reduction the protein becomes active and 02-sensitive. EPR-monitored redox titrations have been carried out of both the resting and the activated enzyme. In the course of a reductive titration, the resting protein becomes activated and begins to produce molecular hydrogen at the expense of reduced titrant. Therefore, equiiibrium potentials are undefined, and previously reported apparent The iron-sulfur protein hydrogenase catalyzes the reversible activation of molecular hydrogen, a process involving the transfer of two electrons. Most presently known hydrogenases are also nickel proteins. The nickel ion is generally assumed to be the redox-active catalytic center [l, 21. A small subclass is formed by the Fe-hydrogenases; these enzymes presumably contain no other potentially redox active transition metals than iron [3]. By exclusion, this implies that the H2 activation is located on an iron-sulfur cluster. Redox catalysis is not Correspondence to W.
Cobalamin-dependent methionine synthase from Escherichia coli catalyzes the last step in de novo methionine biosynthesis. Conversion of the inactive cob(II)alamin form of the enzyme, formed by the occasional oxidation of cob(I)alamin during turnover, to an active methylcobalamin-containing form requires a reductive methylation of the cofactor in which an electron is supplied by reduced flavodoxin and the methyl group is derived from S-adenosyl-L-methionine. E. coli flavodoxin acts specifically in this activation reaction, and neither E. coli ferredoxin nor flavodoxin from the cyanobacterium Synechococcus will substitute, despite their highly similar midpoint potentials for one-electron transfer. As assessed by EPR spectroscopy, the binding of flavodoxin to cob(II)alamin methionine synthase results in a change in the coordination geometry of the cobalt from five-coordinate to four-coordinate. Histidine 759 of methionine synthase, which replaces the normal lower ligand dimethylbenzimidazole on binding of methylcobalamin to methionine synthase, is dissociated from the cobalt of the cobalamin by the binding of flavodoxin. The association of flavodoxin and methionine synthase depends on ionic strength and pH; the pH dependence corresponds to the uptake of one proton on association. The formation of a complex between flavodoxin and methionine synthase perturbs the midpoint potentials of the flavin and cobalamin cofactors only marginally and without any significant thermodynamic advantage for electron transfer to the cobalamin of methionine synthase. No significant binding was seen between oxidized flavodoxin and methylcobalamin methionine synthase. A model for the interaction of methionine synthase with flavodoxin is proposed in which flavodoxin binding leads to changes in the distribution of methionine synthase conformations.
The low-temperature S2-state EPR signal at g = 4 from the oxygen-evolving complex (OEC) of spinach Photosystem-II-enriched membranes is examined at three frequencies, 4 GHz (S-band), 9 GHz (X-band) and 16 GHz (P-band). While no hyperfine structure is observed at 4 GHz, the signal shows little narrowing and may mask underlying hyperfine structure. At 16 GHz, the signal shows g-anisotropy and a shift in g-components. The middle Kramers doublet of a near rhombic S = 5/2 system is found to be the only possible origin consistent with the frequency dependence of the signal. Computer simulations incorporating underlying hyperfine structure from an Mn monomer or dimer are employed to characterize the system. The low zero field splitting (ZFS) of D = 0.43 cm-1 and near rhombicity of E/D = 0.25 lead to the observed X-band g value of 4.1. Treatment with F- or NH3, which compete with Cl- for a binding site, increases the ZFS and rhombicity of the signal. These results indicate that the origin of the OEC signal at g = 4 is either an Mn(II) monomer or a coupled Mn multimer. The likelihood of a multimer is favored over that of a monomer.
In this report, we describe the electron paramagnetic resonance (EPR) spectroscopic characterizations of the fast-relaxing ubisemiquinone (SQ(Nf)) species associated with NADH-ubiquinone oxidoreductase (complex I) detected in tightly coupled submitochondrial particles (SMP). The signals of SQ(Nf) are observed only in the presence of delta muH+, whereas other slowly relaxing SQ species, SQ(Ns) and SQ(Nx), are not sensitive to delta muH+. In this study, we resolved the EPR spectrum of the delta muH+-sensitive SQ(Nf), which was trapped during the steady-state NADH-Q1 oxidoreductase reaction, as the difference between coupled and uncoupled SMP. Thorough analyses of the temperature profile of the resolved SQ(Nf) signals have revealed previously unrecognized spectra from delta muH+-sensitive SQ(Nf) species. This newly detected SQ(Nf) signals are observable only below 25 K, similar to the cluster N2 signals, and exhibit a doublet signal with a peak-to-peak separation (deltaB) of 56 G. In this work, we identify the partner to the interacting cluster N2. We have analyzed the g = 2.00 and g = 2.05 splittings using a computer simulation program that includes both exchange and dipolar interactions as well as the g-strain effect. Computer simulation of these interaction spectra showed that cluster N2 and fast-relaxing SQ(Nf) species undergo a spin-spin interaction, which contains both exchange (55 MHz) and dipolar interaction (16 MHz) with an estimated center-to-center distance of 12 A. This finding delineates an important functional role for this coupled [(N2)(red)-SQ(Nf)] structure in complex I, which is discussed in connection with electron transfer and energy coupling.
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