Vast world reserves of methane gas are underutilized as a feedstock for production of liquid fuels and chemicals due to the lack of economical and sustainable strategies for selective oxidation to methanol1. Current processes to activate the strong C–H bond (104 kcal/mol) in methane require high temperatures, are costly and inefficient, and produce waste2. In nature, methanotrophic bacteria perform this reaction under ambient conditions using metalloenzymes called methane monooxygenases (MMOs). MMOs are thus the optimal inspiration for an efficient, green catalyst3. There are two types of MMOs. Soluble MMO (sMMO), which is expressed by several strains of methanotrophs under copper limited conditions, oxidizes methane with a well characterized catalytic diiron center4. Particulate methane monooxygenase (pMMO), an integral membrane metalloenzyme produced by all methanotrophs, is composed of three subunits, pmoA, pmoB, and pmoC, arranged in a trimeric α3β3γ3 complex5. Despite 20 years of research and the availability of two crystal structures, the metal composition and location of the pMMO metal active site are not known. Here we show that pMMO activity is dependent on copper, not iron, and that the copper active site is located in the soluble domains of the pmoB subunit rather than within the membrane. Recombinant soluble fragments of pmoB (spmoB) bind copper and exhibit propylene and methane oxidation activities. Disruption of each copper center in spmoB by mutagenesis indicates that the active site is a dicopper center. These findings resolve the pMMO controversy and provide a promising new approach to developing environmentally friendly C–H oxidation catalysts.

Ferritins are the main iron storage proteins found in animals, plants, and bacteria. The capacity to store iron in ferritin is essential for life in mammals, but the mechanism by which cytosolic iron is delivered to ferritin is unknown. Human ferritins expressed in yeast contain little iron. Human Poly r(C)-Binding Protein 1 (PCBP1) increased the amount of iron loaded into ferritin when expressed in yeast. PCBP1 bound to ferritin in vivo, and bound iron and facilitated iron loading into ferritin in vitro. Depletion of PCBP1 in human cells inhibited ferritin iron loading and increased cytosolic iron pools. Thus, PCBP1 can function as a cytosolic iron chaperone in the delivery of iron to ferritin.Ferritins are iron storage proteins that are ubiquitously expressed in animals, plants, and bacteria. They serve both to sequester excess iron taken up by the cell and to release stored iron to meet the cell's metabolic needs during iron scarcity (1). In animals, ferritin is a cytosolic heteropolymer consisting of 24 subunits of H-and L-isoforms that assemble into a hollow sphere into which iron is deposited. Ferritin H-chains contain the iron-binding and ferroxidase activities that are required for mineralization of the ferritin core. Deletion of the H-ferritin gene is lethal in mice (2) and in flies (3).In cells, metallochaperones deliver metals to their cognate enzymes and transporters. Although cytosolic copper and nickel chaperones have been described (4-7), no cytosolic iron chaperones have been identified, despite the presence of numerous iron-dependent enzymes in the cytosol. Frataxin, the protein lacking in the neurological disease Friedreich's ataxia, functions as a mitochondrial iron chaperone for iron-sulfur cluster and heme biosynthesis (8,9).Fungi are anomalous among eukaryotes in that they do not express ferritins. We expressed human H-and L-ferritins in the yeast Saccharomyces cerevisiae. The peptides assembled into multimeric complexes with properties similar to native human ferritins, but contained only small amounts of iron ( fig. S1, A and B). We hypothesized that yeast might also lack the requisite iron chaperones needed for delivery of iron to ferritin and designed a genetic screen to identify human genes that, when expressed in yeast, could increase the amount of iron loaded into ferritin. We introduced an iron-regulated FeRE/HIS3 reporter construct (10) into a yeast strain expressing H-and L-ferritin (Fig. 1A). This construct confers histidine prototrophy to cells when the reporter is bound and transcriptionally activated by Aft1p, the major irondependent transcription factor in yeast. Aft1p is activated during periods of cytosolic iron depletion (11), which could occur if substantial amounts of cytosolic iron were diverted into ferritin. †To whom correspondence should be addressed.

After budding, the human immunodeficiency virus (HIV) must 'mature' into an infectious viral particle. Viral maturation requires proteolytic processing of the Gag polyprotein at the matrix-capsid junction, which liberates the capsid (CA) domain to condense from the spherical protein coat of the immature virus into the conical core of the mature virus. We propose that upon proteolysis, the amino-terminal end of the capsid refolds into a β-hairpin/helix structure that is stabilized by formation of a salt bridge between the processed amino-terminus (Pro1) and a highly conserved aspartate residue (Asp51). The refolded amino-terminus then creates a new CA-CA interface that is essential for assembling the condensed conical core. Consistent with this model, we found that recombinant capsid proteins with as few as four matrix residues fused to their aminotermini formed spheres in vitro, but that removing these residues refolded the capsid amino-terminus and redirected protein assembly from spheres to cylinders. Moreover, point mutations throughout the putative CA-CA interface blocked capsid assembly in vitro, core assembly in vivo and viral infectivity. Disruption of the conserved amino-terminal capsid salt bridge also abolished the infectivity of Moloney murine leukemia viral particles, suggesting that lenti-and oncoviruses mature via analogous pathways.

Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. Previous biochemical and structural studies of pMMO have focused on preparations from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. A pMMO from a third organism, Methylocystis species strain M, has been isolated and characterized. Both membrane-bound and solubilized Methylocystis sp. strain M pMMO contain ~2 copper ions per 100 kDa protomer and exhibit copper-dependent propylene epoxidation activity. Spectroscopic data indicate that Methylocystis sp. strain M pMMO contains a mixture of CuI and CuII, of which the latter exhibits two distinct type 2 CuII electron paramagnetic resonance (EPR) signals. Extended X-ray absorption fine structure (EXAFS) data are best fit with a mixture of Cu–O/N and Cu–Cu ligand environments with a Cu–Cu interaction at 2.52–2.64 Å. The crystal structure of Methylocystis sp. strain M pMMO was determined to 2.68 Å resolution and is the best quality pMMO structure obtained to date. It provides a revised model for the pmoA and pmoC subunits and has led to an improved model of M. capsulatus (Bath) pMMO. In these new structures, the intramembrane zinc/copper binding site has a different coordination environment from that in previous models.

Protein unfolding can be induced both by heating and by cooling from ambient temperatures. 1 Accurate analysis of heat and cold denaturation processes has the potential to unveil hitherto obscure aspects of protein stability and dynamics. 2 For instance, while heat denaturation is generally highly cooperative, cold denaturation has been suggested to occur in a noncooperative fashion. 3,4 This view has been recently supported by an NMR study of ubiquitin in reverse micelles at very low temperatures, 5 but this is still controversial since Van Horn et al., 6 on the basis of similar NMR data, and Kitahara et al., 7 by an NMR study at 2 kbar, found a simple two-state behavior for the low-temperature unfolding of ubiquitin.To reach a consensus on this debate and other general issues, it is necessary to investigate cold denaturation further. However, since the cold denaturation of most proteins occurs well below the freezing point of water, full access to the cold denatured state is normally limited for the obvious reason that water freezes at 0 °C. The most common approach to circumvent this difficulty has been to try to raise the temperature of cold denaturation using destabilizing agents such as extreme pH values, chemical denaturants, cryosolvents, or very high pressure. 7-10 Alternatively, some laboratories used proteins destabilized by a combination of point mutations and denaturing agents. 9 The main drawback of these approaches is that it is not generally easy to extrapolate results to physiological conditions. On the other hand, there are methods aimed at keeping water in a supercooled condition, but these studies have also invariably used destabilized proteins. 11,12Following a different approach, we looked for a protein whose cold denaturation could be studied without the need for destabilization in a normal buffer at physiological pH within a temperature range accessible to several techniques. Here we describe the cold and heat denaturation of yeast frataxin (Yfh1) measured both by NMR and CD spectroscopies. In a systematic study of the factors that influence the thermal stability of the frataxin fold, we had previously shown that although they share the same fold, three orthologues from E. coli (CyaY), S. cerevisiae (Yfh1) and H. sapiens (hfra), are characterized, under the same conditions, by a remarkable variation of melting temperatures. 13 Yfh1, the one with lowest heat denaturation temperature, seemed a promising candidate for cold denaturation above 0 °C. Yfh1 and 15 Nlabeled Yfh1 were expressed in E. coli as described by He et al. 14 Since variations of ionic NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript strength lead to significant increases in the melting temperature, we restricted the present investigation to solutions of Yfh1 in salt-free buffers.We recorded 1D and 2D NMR spectra of Yfh1 either in TRIS at pH 7.0 or in HEPES at pH 7.0 in the temperature range −5 to 45 °C. Typically, 0.3-0.5 mM unlabeled or 15 N uniformly labeled protein samples were used. Thanks to t...