Vitamin B12-dependent methionine synthase catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine via the enzyme-bound cofactor methylcobalamin. To carry out this reaction, the enzyme must alternately stabilize six-coordinate methylcobalamin and four-coordinate cob(I)alamin oxidation states. The lower axial ligand to the cobalt in free methylcobalamin is the dimethylbenzimidazole nucleotide substituent of the corrin ring; when methylcobalamin binds to methionine synthase, the ligand is replaced by histidine 759, which in turn is linked by hydrogen bonds to aspartate 757 and thence to serine 810. We have proposed that these residues control the reactivity of the enzyme-bound cofactor both by increasing the coordination strength of the imidazole ligand and by allowing stabilization of cob(I)alamin via protonation of the His-Asp-Ser triad. In this paper we report results of mutation studies focusing on these catalytic residues. We have used visible absorbance spectroscopy and electron paramagnetic resonance spectroscopy to probe the coordination state of the cofactor and have used stopped-flow kinetic measurements to explore the reactivity of each mutant. We show that mutation of histidine 759 blocks turnover, while mutations of aspartate 757 or serine 810 decrease the reactivity of the methylcobalamin cofactor. In contrast, we show that mutations of these same residues increase the rate of AdoMet-dependent reactivation of cob(II)alamin enzyme. We propose that the reaction with AdoMet proceeds via a different transition state than the reactions with homocysteine and methyltetrahydrofolate. These results provide a glimpse at how a protein can control the reactivity of methylcobalamin.

We applied a new phase-modulation technique for nonlinear laser spectroscopy with sub-Hz relative resolving power to measure fundamental relaxation processes of the N-V center in diamond. Complementary EPR experiments versus temperature establish the ground-state spin character in the absence of optical illumination and show that spin-lattice decay occurs via two-phonon processes involving the dominant vibrational mode. The combined results permit deduction of reliable fine-structure assignments for three states of the center and accurate values for zero-field intersystem crossing and spin-lattice relaxation rates from linewidths of individual resonances in the four-wave-mixing spectrum. PACS numbers: 71.55.Ht, 42.65.Ma, 76.30.Mi, 78.50.Ec Nearly degenerate four-wave-mixing (NDFWM) spectroscopy with 1-Hz resolution based on acousto-optic frequency-modulation techniques has previously been applied to frequency-domain measurements of slow relaxation processes in impurity-doped solids [1] and pointdefect systems [2]. This coherent spectroscopy has proven very useful for precise measurements of decay processes too slow for eA'ective signal averaging in real time.Here we introduce a new optical technique for the performance of NDFWM with much higher resolution (10 mHz), and apply it in concert with double-cavity EPR experiments to resolve the controversy over the energylevel scheme of the N-V center in diamond and to study system dynamics.We provide evidence for a hypothesized metastable state and demonstrate that precise measurements of intersystem crossing and spin-lattice relaxation rates can be obtained from linewidth measurements of individual, ultranarrow resonances in the fourwave-mixing spectrum recorded with sub-Hz resolution.The N-V center is a product of irradiation and annealing processes in diamond crystals containing nitrogen [3] and exhibits a zero-phonon line at 637 nm assigned by uniaxial stress measurements[4] to an 2 E electricdipole transition at a site of trigonal symmetry. It consists of substitutional-nitrogen-vacancy pairs oriented along equivalent (111) directions, and exhibits triplet spin resonance which was first attributed to a metastable excited state [5] because it required optical illumination in the N-V absorption band. This conclusion and even the existence of a metastable state was challenged recently in a series of papers reporting hole burning [6], optically detected magnetic resonance [7], and Raman heterodyne experiments [g], which indirectly suggested that the triplet state occurs in the ground-state manifold rather than the metastable manifold.However, these experiments were performed with optical illumination of the centers, and are consistent with alternative explanations based either on absorption at 637 nm by a metastable excited triplet population or on a third possible energy-level structure of the center. To examine these possibilities, the current work was undertaken to determine fine-3A 1E 'A -'E= I» nm g (1a& b) I 'A (c) FIG. I. Possible energy level schemes of the N-...

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.

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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.