SCIENCE/TECHNOLOGY "This is the first mitochondrial super oxide dismutase that has been done, however." Tainer lists three of the most interest ing structural features discovered in the mitochondrial Mn-SOD: First, at the lev el of the individual subunits, the N-terminal domain—amino acid residues 1 through 84—is "formed primarily by two long antiparallel α-helices separated by a tight turn, forming a helical hairpin structure." The C-terminal domain—res idues 85-198—contains five α-helices and a three-stranded β sheet. The activesite manganese joins the two domains and is positioned between the N-terminal α-helices and C-terminal β sheet, Tainer says. Second, at the level of the dimers that make up the tetrameric enzyme, Tainer and his collaborators discovered that each active site is made up of residues from both subunits of the dimer. That is, al though each manganese ion is bound within a subunit, residues from both subunits contribute to the metal binding site. And third, the tetrameric structure of Mn-SOD is stabilized by interactions between the helical hairpins. The heli cal hairpins of two subunits assemble to form a "four-helix bundle" with a left-handed twist, and the helical hair pins of the other two subunits assem ble to form another four-helix bundle on the opposite side of the enzyme. Tainer points out that the four-helix bundle interfaces in Mn-SOD appear to be important not only for the enzyme's assembly, but also for its stability and activity. In the subunit, if the helical hairpin is not tethered by formation of the four-helix bundle, the two ligands contributed from the hairpin helices to the metal binding site are destabilized, adversely affecting the manganese ac tive-site geometry and the enzyme's ac tivity, Tainer says. It is the existence of Mn-SODs contain ing destabilized four-helix bundles that caught the attention of Tainer and his co workers. Other researchers had shown that the sequence of the helical hairpin is highly conserved in mitochondrial MnSODs from different species. Yet human Mn-SOD is polymorphic, Tainer says, and one naturally CHzajirring variant, in which a hydrophobic isoleucine in the helical hairpin is replaced with a hydrophilic threonine, has been found in two of six complementary DNA libraries examined by the Scripps researchers. That single amino acid change is far 20
OCTOBER 26,1992 C&EN
from insignificant, Tainer stresses. ''Within the four-helix bundle interface, that isoleucine is the largest contributor to buried surface area," he says. Replace ment of this isoleucine with threonine likely would destabilize the bundle, The tightening of environmental laws leading to the observed decrease in en and practices has meant that chemical zyme activity. Together with Tainer, companies and oil refiners are finding it Hickey, and Hallewell, Scripps research increasingly difficult to get rid of ers Maurice Boissinot and Michael J. "spent" catalysts, mainly because of the Johnson are working with Diane E. Ca- potentially harmful metals they contain. belli of Brookhaven National Laboratory But now that Metrex is operating a plant and James R. Lepock of the University of in Heerlen, the Netherlands, that breaks Waterloo, Canada, to complete a com down catalysts into their components, prehensive characterization of the stabil hope is at hand for some alleviation of ity and assembly of this mutant. the problem. "The company name is coined from The mystery is: Why should such a change be preserved in humans? Mito the process, which entails metals recovchondria consume more than 90% of the ery by extraction," notes Metrex director cell's oxygen, and the mitochondrial res Wladimir Kesber. "It says what we're piratory chain is the source of a large doing, and can be pronounced fairly easflux of oxygen radicals. Mitochondrial ily in most languages. Ours is the only DNA is particularly susceptible to oxida operation of its kind anywhere. It could tive damage because of this exposure to be the forerunner of similar units as deoxygen as well as relatively inefficient mand for our service grows. In a sense, DNA repair mechanisms and the lack of we are using the Heerlen operation as a demonstration plant." histone proteins, Tainer points out. Adds Hans S. M. Jocker, Metrex genThe solution may lie in the immune system's apparent use of oxygen radi eral manager, who cofounded the comcals to eliminate cells infected with pany with Kesber: "We recover the metpathogens. Other researchers have als and the alumina usually used as carshown, for example, that the ability of rier, and sell the products to companies tumor necrosis factor (TNF) to kill vi that can use them. We don't regenerate rus-infected cells is inhibited by SOD. the catalysts as such." And stimulated neutrophils and mac The two partners' work experience rophages, two types of immune cells makes them ideally suited for the task at responsible for eliminating pathogens, hand. Jocker has had close dealings with exhibit a characteristic "respiratory refinery and petrochemical operations burst" in which superoxide is generat through his service company, Reakt, ed at a membrane. headquartered near Cambridge, EnThus, Tainer suggests, Mn-SOD vari gland. Kesber, who has been associated ants may exhibit a balanced polymor with a number of international chemical phism analogous to that of human he companies over the years, now has his moglobin, in which a structurally and own chemicals recovery business called functionally defective single-site mu Chemconserve, based at Rijswijk, on the tant—sickle cell hemoglobin—is main outskirts of The Hague. To raise the cash tained in the population owing to a com needed to build the plant and start the pensatory advantage—malarial resis business, the Metrex shareholding has tance—conferred on the hétérozygote. been extended to include four financial The advantage conferred by destabilized concerns. Mn-SOD could be greater resistance to The $15 million facility is located in infections in general. The downside, a the southeast corner of the Netherlands severe one, is greater susceptibility to close to the Belgian and German bordegenerative diseases, many associated ders. It can process up to 15 million lb a with old age, that appear to be caused year of hydrodesulfurization catalysts by oxidative damage to cells. that have reached the end of their useful Tainer says a great deal of work needs life. But it is designed to take double that to be done to test this hypothesis, and he quantity without much difficulty, or hopes other chemists will become in- even more as demand requires. Cobalt, volved. "There are a lot of mysteries that molybdenum, and nickel contained in the original catalyst formulation, along deserve attention," he says. Rudy Baum with vanadium adsorbed during the
Facility recovers metals from spent catalyst
processing of the oil fractions, are recovered. Other products stemming from the operation are aluminum oxide, which goes to make ceramic and refractory bricks and tiles, and gypsum, from the flue gas. "We can justifiably claim that we fully recover the materials for reuse," Jocker maintains. "We have the unique capability of being able to achieve a full metals extraction." Although the operation currently centers on hydrodesulfurization catalysts, he and Kesber foresee its eventual extension into use with other spent catalyst systems. During the 1960s, the need to remove sulfur from crude oil and from refinery fractions spurred the development of hydrodesulfurization catalysts. But during use, deposits of carbon, hydrocarbons, and sulfur build up on the catalysts7 surface, clogging the pores of the aluminum oxide carrier and thereby reducing the catalysts7 ability to function. Although they can be revitalized by subjection to various processes, there is a limit to the number of times regeneration is practicable before the structure becomes permanently destroyed or the catalysts become poisoned. Some 25 years ago, when the catalysts reached the end of their useful life, it was the practice for oil refining companies to burn off the entrained carbon, sulfur, and hydrocarbons before selling the material to iron and steel producers as a relatively inexpensive source of cobalt, molybdenum, and nickel, Jocker recalls. But this outlet tailed off in the 1970s when refiners stopped the stripping step because of the increasing costs involved. "Steel makers don't like sulfur because of the problems it causes," Jocker observes. 'They do like carbon, but not
in the form in which it occurs in the spent catalysts. Yet another problem that arose in the late 1970s was that in some cases phosphorus was added to enhance catalytic capability. And steel and phosphorus don't go well together because phosphorus makes steel brittle." As a result, the untreated spent catalysts were no longer of value to the smelters. So instead of being usable wastes, they ended up being dumped in landfills. That practice, however, was almost universally banned by the mid-1980s. "Some are looking into the possibility of encapsulating spent catalysts before sending them to landfills," Jocker remarks. "We see this as a short-term solution." There is no shortage of material. As a rule of thumb, Jocker points out, some 10 million lb of hydrodesulfurization catalysts are in use annually throughout Western Europe. The same amount is regenerated by service companies, while a similar quantity builds up each year as unusable waste. Following a thorough review, Jocker and Kesber came to realize that the dilemma the oil refiners faced presented a potentially attractive business opportunity provided a viable method for extracting and recovering the metals could be developed. Toward this end, they sought the help of TNO, the Netherlands state-sponsored organization for applied scientific research. "There is nothing particularly new in what we eventually came up with," says Ruud Gerritsen, who directed the development of the process at TNO before joining Metrex as technology manager. "For instance, the solvent extraction method for separating molybdenum is similar to that used for recovering the element from ura-
Metrex plant in Heerlen, the Netherlands
nium ores. What makes the process unique is the way the different steps of the system have been linked together." Following successful pilot plant trials at TNCs Apeldoorn facilities, some of the equipment was transported and refitted at the Heerlen site. The composition of used catalyst that arrives at the plant varies somewhat from one delivery to another. So the catalysts are analyzed and graded by their makeup. When a batch has been selected for putting through the system, it goes first to a vibrating sieve to separate unwanted debris. It then passes to a fluidizedbed combustion chamber and on to a rotary tube furnace, both maintained at temperatures up to 850 °C. The spent catalyst charge is pulverized in the fluidized bed to a particle size of less than a millimeter. Air injection during the heating process converts entrained carbon, hydrocarbons, and sulfur mainly to carbon dioxide and sulfur dioxide, that then move out with the flue gas. At the same time, metal sulfides become oxidized to their corresponding sulfates. After cooling, these are dissolved in a dilute sulfuric acid leaching step, leaving behind most of the aluminum oxide and silica, if present in the original catalyst charge, as insoluble residue. The acid solution of the metal sulfates goes through a series of extraction steps with various organic solvents. From the fractions, cobalt and nickel are recovered as sulfates, vanadium is converted to ammonium vanadate, and molybdenum is extracted as ammonium molybdate or recovered as molybdenum oxide from the flue gas. Meanwhile, the alumina residue, recovered by vacuum filtration, is washed and dried, and the flue gas passes through lime, which converts the sulfur oxides to calcium sulfate, or gypsum. The sulfuric acid is regenerated and recycled. The metal content of hydrodesulfurization catalysts typically is 2 to 3% by weight cobalt, about 4% nickel, and up to 8% molybdenum. In broad terms, about half the weight of spent catalyst consists of alumina. The combined carbon, hydrocarbons, and sulfur account for some 35% of the weight. The remaining 15% or so represents cobalt, molybdenum, nickel, and vanadium sulfides. The Metrex process results in a metals recovery efficiency that exceeds 90%. Dermot O'Sullivan OCTOBER 26,1992 C&EN
21
