Ultraviolet resonance Raman spectroscopy reveals that, when heated at pH 3, a substantial fraction (30 %) of cytochrome c converts to a β-sheet structure, at the expense of turns and helices. β-sheet formation is rapid, exhibiting a 2 µs rise time, following a temperature jump. It is proposed that a short β-sheet segment, comprised of residues 37-39 and 58-61 extends itself into the large 37-61 loop, when the latter is destabilized by protonation of H27, which forms an anchoring H-bond to loop residue P44. This conformation change ruptures the Met80-Fe bond, as revealed by changes in ligation-sensitive heme-resonant Raman bands. It also induces peroxidase activity with the same temperature profile. This process is suggested to model the apoptotic peroxidation of cardiolipin by cytochrome c.We report resonance Raman (RR) spectroscopic evidence for a hitherto unrecognized conformational transition to β-sheet structure in cytochrome c (cyt c), which may have important functional consequences.In addition to its electron-transfer activity in mitochondria, cyt c plays a key role in apoptosis, 1 and partial unfolding seems to be a critical element in the mechanism. Jemmerson et al. have observed that, in association with lipid vesicles, cyt c binds an antibody that recognizes an unfolded region around residue Pro44, and that the same response is seen in apoptotic cells. 2 Belikova et al. report that binding to cardiolipin induces peroxidase activity in cyt c, producing cardiolipin hydroperoxides that are required for release of pro-apoptotic factors. 3 It seems likely that this activity is triggered by changes in heme ligation when cyt c interacts with lipids. 4 In addition to cardiolipin, 5 oleic acid 6 has been observed to destabilize the cyt c fold; the oleic acid effect can be partially reversed by ATP, a component of the apoptosome. 6We find that heating cyt c under destabilizing conditions (pH 3) not only unfolds a significant fraction of the protein, but converts it to a β-sheet structure. This conversion is reversible unless the concentration exceeds 50 µM, when cyt c precipitates upon heating. Moreover β-sheet formation is remarkably fast, occurring on the microsecond time scale. This structural change is associated with rupture of the Fe-S bond (heme-Met80), and induction of peroxidase activity.The evidence for β-sheet formation comes from UVRR spectroscopy. Excitation at 197 nm produces optimal enhancement of amide vibrational modes, whose frequencies and intensities are diagnostic of secondary structure. 7a UVRR signatures have been extracted from a suite of structurally characterized proteins, allowing quantitation of secondary structure. 7b Figure 1 spiro@princeton.edu. shows the result of applying this procedure (supporting info, S1) to UVRR spectra of equine Fe III cyt c at pH 3. At 20°C, the secondary structure is 18 % β-turn, 40 % α-helix, and 42 % unordered structure. This composition is consistent with the crystal structure of native cyt c 8 . NIH Public AccessAs the temperature rises, al...

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MnO2 nanoparticles, similar to those found in soils and sediments, have been characterized via their UV–visible and Raman spectra, combined with dynamic light scattering and reactivity measurements. Synthetic colloids were prepared by thiosulfate reduction of permanganate, their sizes controlled with adsorbates acting as capping agents: bicarbonate, phosphate, and pyrophosphate. Biogenic colloids, products of the manganese oxidase, Mnx, were similarly characterized. The band-gap energies of the colloids were found to increase with decreasing hydrodynamic diameter, D h, and were proportional to 1/D h 2, as predicted from quantum confinement theory. The intensity ratio of the two prominent Mn–O stretching Raman bands also varied with particle size, consistent with the ratio of edge to bulk Mn atoms. Reactivity of the synthetic colloids toward reduction by Mn2+, in the presence of pyrophosphate to trap the Mn3+ product, was proportional to the surface to volume ratio, but showed surprising complexity. There was also a remnant unreactive fraction, likely attributable to Mn­(III)-induced surface passivation. The band gap was similar for biogenic and synthetic colloids of similar size, but decreased when the enzyme solution contained pyrophosphate, which traps the intermediate Mn­(III) and slows MnO2 growth. The band gap/size correlation was used to analyze the growth of the enzymatically produced MnO2 oxides.

Global cycling of environmental manganese requires catalysis by bacteria and fungi for MnO2 formation, since abiotic Mn(II) oxidation is slow under ambient conditions. Genetic evidence from several bacteria implicates multicopper oxidases (MCOs) as being required for MnO2 formation. However, MCOs catalyze one-electron oxidations, whereas conversion of Mn(II) to MnO2 is a two-electron process. Trapping experiments with pyrophosphate (PP), a Mn(III) chelator, have demonstrated that Mn(III) is an intermediate in Mn(II) oxidation when mediated by exosporium from the Mn-oxidizing bacterium Bacillus SG-1. The reaction of Mn(II) depends on O2 and is inhibited by azide, consistent with MCO catalysis. We show that the subsequent conversion of Mn(III) to MnO2 also depends on O2 and is inhibited by azide. Thus, both oxidation steps appear to be MCO-mediated, likely by the same enzyme, indicated by genetic evidence to be the MnxG gene product. We propose a model of how the manganese oxidase active site may be organized to couple successive electron transfers to the formation of polynuclear Mn(IV) complexes as precursors to MnO2 formation.