Natarajan Ravi

[20] Use of rapid kinetics methods to study the assembly of the diferric-tyrosyl radical cofactor of E. coli ribonucleotide reductase

The SF-Abs, RFQ-EPR, and RFQ-Möss data on the R2 reconstitution reaction are all consistent with the mechanism of Scheme I, in which the intermediate X is the immediate precursor to the product cofactor, and illustrate how the continuous SF approach and the discontinuous RFQ methods can be complementary. Given the inherent differences in the methods, it should not be taken for granted that data from the two will be consistent. A number of problems can be associated with the RFQ approach. For example, isopentane could conceivably interfere with or alter the chemistry to be studied. A second potential problem involves temperature-dependent equilibria among different intermediate species. This problem has been encountered by Dooley et al. with the 6-hydroxydopa-requiring protein, plasma amine oxidase and was previously observed with the adenosylcobalamin-dependent ribonucleotide reductase by Blakley and co-workers. This potential complication should be considered when discrepancies arise between SF and RFQ data and in low temperature structural studies of reactive intermediates in general. Each of the three methods employed can yield time-resolved quantitation of reaction components. In this regard, SF-Abs has the disadvantage of poor resolution, such that quantitation of individual components most often requires sophisticated mathematical analysis. Obvious advantages to the RFQ-Möss method are the presence of an internal standard (the known amount of 57Fe being proportional to the total absorption area) and the spectroscopic activity of all reaction components which contain iron. In our hands, quantitation by RFQ-EPR was most problematic and least reproducible. This irreproducibility most likely relates to heterogeneity among samples in terms of volume and density. As discussed in detail by Ballou and Palmer, the packing factor, which relates to the fraction of a sample made up by the reaction solution (the remainder being frozen isopentane), is dependent on the investigator. Given this caveat, it is not surprising that the RFQ-EPR data had the greatest uncertainty in our hands. Placing a chemically unreactive, EPR active standard in each reaction mixture could help alleviate this problem. Time-resolved Möss methods can be extremely powerful if excellent, nonoverlapping reference spectra of starting materials, products, and intermediates are available. All of the iron centers can be examined simultaneously. The problems associated with Möss arise from its extreme insensitivity. It takes millimolar solutions of proteins and several days for data collection of each time point.(ABSTRACT TRUNCATED AT 400 WORDS)

Mössbauer Characterization of Paracoccus denitrificans Cytochrome c Peroxidase FURTHER EVIDENCE FOR REDOX AND CALCIUM BINDING-INDUCED HEME-HEME INTERACTION

Mössbauer and electron paramagnetic resonance (EPR) spectroscopies were used to characterize the diheme cytochrome c peroxidase from Paracoccus denitrificans (L.M.D. 52.44). The spectra of the oxidized enzyme show two distinct spectral components characteristic of low spin ferric hemes (S = 1/2), revealing different heme environments for the two heme groups. The Paracoccus peroxidase can be non-physiologically reduced by ascorbate. Mössbauer investigation of the ascorbate-reduced peroxidase shows that only one heme (the high potential heme) is reduced and that the reduced heme is diamagnetic (S = 0). The other heme (the low potential heme) remains oxidized, indicating that the enzyme is in a mixed valence, half-reduced state. The EPR spectrum of the half-reduced peroxidase, however, shows two low spin ferric species with gmax = 2.89 (species I) and gmax = 2.78 (species II). This EPR observation, together with the Mössbauer result, suggests that both species are arising from the low potential heme. More interestingly, the spectroscopic properties of these two species are distinct from that of the low potential heme in the oxidized enzyme, providing evidence for heme-heme interaction induced by the reduction of the high potential heme. Addition of calcium ions to the half-reduced enzyme converts species II to species I. Since calcium has been found to promote peroxidase activity, species I may represent the active form of the peroxidatic heme.

Characterization of three proteins containing multiple iron sites: rubrerythrin, desulfoferrodoxin, and a protein containing a six-iron cluster.

Publisher Summary This chapter focuses on three proteins: (1) rubrerythrin (Rr), (2) desulfoferrodoxin (Dfx), and (3) an [Fe—S] protein containing a six-iron cluster. These three proteins display the presence of redox metal centers with metal compositions spanning from mono- through bi- to hexanuclearity. Rr was first characterized from Desulfovibrio vulgaris (Hildenborough) (DvH). All of its purification procedures are performed at 4 and pH 7.6. The presence of Rr is judged by a change in absorbance at 490 nm after ascorbate reduction. Rr is composed of two identical subunits of molecular mass (MM) 21.9 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optical, electron paramagnetic resonance (EPR), and Mossbauer properties show that two of the iron atoms belong to FeS 4 centers similar to Rd-type centers and the other two belong to an exchange-coupled binuclear center. Dfx is a monomer of molecular mass 14 kDa. The iron determination shows that this protein contains about two iron atoms per molecule. The optical, EPR, and Mossbauer properties show that these two iron sites are inequivalent.

Redox and Spin-State Control of the Activity of a Diheme Cytochrome C Peroxidase — Spectroscopic studies

Hydrogen peroxide formed in cells, as the result of incomplete reduction of oxygen, can be removed essentially by two ways: by peroxidases in a process of reduction to water or by catalase in a dismutation reaction. The actions of these enzymes are essential to prevent the accumulation of hydrogen peroxide, diminishing the risk of peroxide-induced damage of cell constituents [1].

Rubrerythrin: A non-heme iron protein with structural analogies to ribonucleotide reductase.

Rubrerythrin, a contraction of rubredoxin and hemerythrin, is the trivial name given to a non-heme iron protein isolated from Desulfovibrio vulgaris (Hildenborough). This protein, whose physiological function is unknown, was first characterized by J. LeGall et al. [(1988) Biochemistry 28, 1636] as being a homodimer of subunit M(r) = 21,900 with four Fe per homodimer distributed as two rubredoxin-type FeS4 centers and one hemerythrin-type diiron cluster. Subsequent analysis of the amino acid sequence of the rubrerythrin gene [Kurtz, D. M., Jr., & Prickril, B.C. (1991) Biochem. Biophys. Res. Commun. 181, 137] revealed an internal homology which suggested that each subunit can accommodate one diiron cluster. Here, we report a procedure for reconstitution of the as-isolated D. vulgaris rubrerythrin with 57Fe. The reconstituted protein was characterized by optical, electron paramagnetic resonance, and Mössbauer spectroscopies. The results indicate successful incorporation of 57Fe into the two types of sites and strongly suggest that each subunit of rubrerythrin can indeed accommodate one diiron cluster as well as one rubredoxin-type center. Combined with amino acid sequence analysis, the spectroscopic characterization further suggests that the rubrerythrin subunit contains a diiron site whose structure is more closely related to that in ribonucleotide reductase than to that in hemerythrin.