SCIENCE & TECHNOLOGY BINUCLEAR Darensbourg (left) and Hall collaborate on computational models of the active site of iron-only hydrogenase.
CAN WE EXPLOIT HYDROGENASES? Insights into nature's substitute for platinum may lead to the design of inexpensive catalysts MICHAEL FREEMANTLE, C&EN LOND Ν
M
ICROORGANISMS EMPLOY
highly complex chemical sys tems to carry out a number of reactions of interest to chemists. The organisms do so with such seeming ease that it's enough to turn chemists who are trying to dupli cate those reactions green with envy Recent research has focused on one such simple chemical process—the reversible oxidation of hydrogen: H 2 ^2H + + 2eThe systems are enzymes known as hydrogenases. Almost all of them rely on abundant metals for their catalytic activi ty For example, nature uses iron-contain ing hydrogenases to catalyze hydrogen production and hydrogenases containing both iron and nickel to catalyze hydrogen consumption. "Microorganisms have been using hy drogen as a primary fuel source for billions of years and have solved the problem of converting hydrogen to electricity by means of the hydrogenases," explains Richard Cammack, professor ofplant biochemistry at King's College London. "Many microorganisms contain more than one type of hydrogenase, each with a dif ferent function. These include hydrogen production in fermentation, hydrogen con sumption for formation of biomass, and HTTP://PUBS.ACS.ORG/CEN
hydrogen oxidation for respiratory ener gy production." Industry, on the other hand, employs precious metals such as platinum, a scarce and nonrenewable resource, to catalyze the generation of electricity from hydro gen using fuel cells and the production of hydrogen from water by electrolysis. "If we can understand how hydroge nases work at a molecular level, we might be able to exploit this knowledge to create cheaper catalysts," Cammack says. Marcetta Y Darensbourg, professor of inorganic chemistry at Texas A&M Uni versity, College Station, points out that chemists use the noble metals iridium, plat inum, and rhodium to catalyze two-elec tron processes such as making and break ing H - H and C - H bonds. "In hydrogenases, nature has taken read ily available nickel and iron, coupled them with thiolato ligands, which are chemical ly versatile sulfur donors, and, over the course of billions ofyears of evolution, as sembled a machine that controls H 2 ac cess, activates H 2 under exceedingly mild conditions, and directs the electrons of the H - H bond through a true molecular wire onto their next job," she says. "This ma chinery at the active site produces asym metric coordination spheres and torqued coordination geometries. The resulting re-
active moieties are difficult to reproduce by synthetic chemists, even when they are guided by crystallographers and spectroscopists on how the atoms must be con nected," she adds. Hydrogenases fall broadly into two ma jor classes defined by their active sites—iron only and nickel-iron. A nonmetal hydroge nase that occurs in methane-generating bacteria is also known. Although that en zyme contains no transition-metal ions, a cofactor of unknown composition has been detected. Cammack points out that active sites of hydrogenases have proven to be some of the most complex and ingenious bioinorganic structures known. Ά strik ing feature is the presence of cyanide and carbonyl ligands to the iron atoms," he ob serves. "While such ligands are common place in inorganic chemistry, they had nev er been seen in enzymes before. They appear to be the key to the very high cat alytic efficiency of hydrogenases." The {NiFe}- and [Fel-hydrogenases have a number of important features in com mon even though they have evolved inde pendently and are genetically unrelated. For example, the crystal structures ofboth classes of hydrogenases reveal proton and hydrogen access and egress routes into the hydrogen-producing and -uptake sites that are deeply buried in the protein in order to prevent oxygen damage. The active sites are connected to the proteins by cysteines—sulftir-containing amino acids. Seminal work on these structural eluci dations was carried out several years ago by groups led by X-ray crystallographer Juan C. Fontecilla-Camps at the Institute of Structural Biology, Grenoble, France, and John W. Peters, associate professor of bio chemistry and protein crystallography at Utah State University "THE CRYSTAL structures show that these active sites are also 'wired' to electron ac ceptor/donor proteins through FeS chains, primarily 4Fe4S clusters," Darensbourg explains. "Most amazingly, crystallography shows that the active sites are discrete bimetallic moieties and distinctly organometalliclike." A group led by Yoshiki Higuchi, pro fessor at Himeji Institute of Technology, Japan, is using X-ray structure analysis, mass spectrometry, and other techniques to determine the structure of the active C & E N / JULY 2 2 , 2002
35
SCIENCE & TECHNOLOGY
rr
~T H+ ,
((CH 3 ) 3 P)(OC) 2 Fe^—^Fe(CO) 2 (CN)
rr ( ( C H 3 ) 3 P ) ( O C ] 2 F e ^ - — Fe(C0)2(CN]
Iron carbonyl thiolate
+ 2e", H, evolution
rr
τ
((CH 3 ) 3 P)(OC) 2 Fe^- - - - Fe(C0)2(CNH) SOURCE: J. Am. Chem. Soc, 123, 9476 (2001)
BIOMIMETIC Analog of iron-only hydrogenase active site catalyzes reduction of protons to dihydrogen, beginning with protonation of Fe-Fe bond. sites of {NiFe]-hydrogenases and to find out which atoms are responsible for acti vating molecular hydrogen. 'Although many crystal structures of the {NiFe] active site are now available, there is still uncertainty about the structure," he tells C&EN. "For example, the iron atom has four nonprotein ligands in the oxidized form. Three of these are diatomic ligands, and one is monatomic or its hydride." Two years ago, Higuchi and coworkers presented mass spectroscopic evidence that SO, a ligand that had never previous ly been reported in biomolecules, is pres ent in the active site of a [NiFe}-hydrogenase obtained from a bacterium known as Desulfovibrio vulgaris Miyazaki \J. Inorg. Biochem., 8 0 , 2 0 5 (2000)}. The group al so reported that bands in the Fourier trans form infrared spectrum of the hydroge nase are ascribable to CO and C N ligands. "We showed that SO, CO, or C N could be candidates for diatomic ligands and S or SH for the monatomic ligands," Higuchi says. "Other groups claim that the diatomic ligands should be C O or C N and the monatomic ligand maybe Ο or OH. How ever, we showed that when our {NiFe}-hydrogenase is activated—that is, reduced by hydrogen—it always liberates hydrogen sulfide." HYDROGEN ACTIVATION in most {NiFe}hydrogenases is reversibly inhibited by oxygen and carbon monoxide. Higuchi's group solved 10 crystal structures to de termine the exact site to which the com petitive inhibitor carbon monoxide re versibly coordinates in a CO-bound {NiFe}-hydrogenase. The research group showed that carbon monoxide is coordi
nated to the Ni atom and not the Fe atom. At the University of Oxford, a group led by professor of chemistry Fraser A. Armstrong is using an electrochemical technique called protein film voltammetry to investigate the active sites of hydrogenases. "Our group specializes in looking at the properties and reactions of redox centers in hydrogenases, and other enzymes and proteins," he tells C&EN. "We have pro tein molecules docked on an electrode so that their activities can be driven directly by our electrochemical instrument." Armstrong's group is using the voltammetry technique to study the activity of a {NiFe}-hydrogenase from the bacterium Allochromatium vinosum. "This enzyme, and oth ers in its class, are quite well characterized," Armstrong remarks. "There is a lot of knowledge from spec troscopy about different states, and some good crys tal structures of inactive, resting forms. However, the whole important subject of the mechanism is quite Armstrong overwhelming and seemingly intractable. So it presents a wonder ful challenge." The group members, working in col laboration with Simon P. J. Albracht, as sociate professor of biochemistry at the University of Amsterdam, recently com pared the hydrogen oxidation activity of the hydrogenase on a graphite electrode with that ofplatinum deposited on an identical electrode. They showed that the
[NiFe}-active site of the enzyme catalyzes the oxidation at rates comparable to those ofplatinum [Chem. Commun., 2002,866}. "Our paper shows not that the activity of the enzyme is comparable to that of plat inum but that the activity of the {NiFe}-site is," Armstrong says. "We also showed, in earlier work, that the hydrogenase is rela tively immune to carbon monoxide poi soning compared with platinum." Armstrong points out, however, that transferring electrons from the active site of the hydrogenase to an electrode can be a problem. "Unlike platinum centers on an electrode, the [NiFe}-site is buried, and some energy is spent driving the electrons across the protein and interface," he ex plains. "If this problem can be remedied and the enzyme film stabilized to run for long periods of time, then platinum may ul timately be replaced by enzymes that can be produced in large quantities at low cost. Improving the lifetime of the enzyme is within the grasp of genetic engineering, and it should be possible to increase the rate of electron transfer with chemical methods. "The paper also clearly shows that it must be possible to make fully functional catalysts from base metals like nickel and iron that challenge the high station of plat inum as a fuel-cell catalyst," he adds. According to Darensbourg, the paper describes an experiment that cried out to be done. "The com bination of electrochem istry and the metalloprotein imbedded in the electrode film illustrates a feasible direction for research to proceed," she says. Darensbourg's group at Texas A & M has b e e n studying organometallic complexes that have compositional and structural similarities to the active site of iron-only hydrogenases. The site contains a cluster of six iron atoms known as the H-cluster with a center consisting of two iron atoms bridged by two sulfur atoms linked by another bridge. "We cannot make an exact model of that active site, but we might learn from retaining the central 2Fe2S core and ex ploring what it takes to get hydrogen
Microorganisms have solved the problem of converting hydrogen to electricity by means of the hydrogenases/' 36
C & E N / JULY 2 2 , 2002
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Switching Green Alga Hydrogen Production On And Off • h e r e is no doubt that biological hydrogen production works and is clean and renewable—though much improvement will be needed before biohydrogen can compete economically as an energy source with fossil fuels or other technological solutions such as silicon solar cells and electrolysis of water," remarks Richard Cammack, professor of plant biochemistry at King's College London. "The ideal is to produce hydrogen in the light. This process has been observed in green algae and cyanobacteria under certain growth conditions."
T:
According to Anastasios Melis, professor of enzymology at the University of California, Berkeley, modification of photosynthesis in unicellular green algae will permit the generation of hydrogen gas as a clean, renewable, and economically viable fuel. Melis and colleagues are developing a process to maximize the solar energy-tohydrogen conversion efficiency of the green alga Chiamydomonas reinhardtii under mass culture conditions. One of the specific objectives of their current work is to improve the stability and func-
RENEWABLE Hydrogen bubbles form inside flask of sulfur-deprived green algae. tionality of [Fe]-hydrogenase, the enzyme that catalyzes hydrogen production in the green algae. Under physiological conditions, the photosynthetic activity of the enzyme is transient, lasting from several seconds to a few minutes, and hydrogen is produced in only small quantities. "The light-dependent oxidation of wa-
38
C & E N / JULY 2 2 , 2 0 0 2
ter results in the release of molecular oxygen," Melis explains. "Oxygen is a positive suppressor of hydrogenase gene expression and a powerful inhibitor of [Fe]-hydrogenase activity." Melis and coworkers have shown that, when they deprive green algae of sulfur nutrients, oxygen production drops below that needed for respiration. In sealed cultures, the sulfur-deprived algae consequently become anaerobic and spontaneously induce a hydrogenase pathway to produce hydrogen photosynthetically. The process continues for 80 hours or so in the light, during which time the cells consume significant amounts of internal starch and protein. The process is reversible, however. In the presence of sulfur, the green algae carry out normal photosynthesis—that is, water oxidation, oxygen evolution, and biomass accumulation. By using the presence and absence of sulfur as a switch, Melis and colleagues cause the algae to alternate between oxygen and hydrogen production and therefore avoid the inhibitive action of oxygen. The two-stage process, which is currently at a development stage in the laboratory, generates an average of 3 ml_ of hydrogen per hour per liter of culture for up to four days. The culture is then returned to normal photosynthesis for two or three days until it is ready to be tapped for hydrogen again. "With current technology, the green alga hydrogen production process is discontinuous, or intermittent," Melis tells C&EN. "Thus, following the period of hydrogen production, green algae need to go back to normal photosynthesis to recover their endogenous substratestarch and protein—that was consumed during hydrogen production. "Overcoming this discontinuity is the top priority in our lab," he continues. "Additional challenges that must be successfully addressed by the biohydrogen community include the design of efficient and low-cost photo-bioreactors, the question of utilizing genetically modified green algae under mass culture (commercial) conditions, and the need to form a working relationship between the alga biotechnology and the energy sector industries."
uptake and activation," Darensbourg says. Last year, Darensbourg's group described the use of nuclear magnetic resonance spectroscopy and hydrogen/deuterium exchange reactions of dinuclear iron thiolate model compounds to investigate the reactivity of Fe(D-Fe(D and Fe(II)-H-Fe(II) complexes \J.Am. Chem. Soc, 123,9710 (2001)}. "WE SHOW that by using phosphine ligands as mimics of cyanide, we can generate a binuclear iron complex that performs an activity assay that is consistent with the chemical characteristics and activity of the enzymes," Darensbourg explains. irWe confirm the necessity of two iron atoms to do the work of one noble metal atom. The models do amazingly well at some of the reactions of hydrogenases." Darensbourg's colleague at Texas A&M, chemistry professor Michael B. Hall, is using computational chemistry to understand the electronic structures and bonding of the active sites of hydrogenases. "Our group has the most complete set of computational models for the active site states of these enzymes," he tells C&EN. "By using synthetic inorganic model compounds to calibrate the theoretical modeling, we have gained confidence that we are producing accurate electronic models for the active sites. The model compounds synthesized by Darensbourg and her group played a key role in our successful modeling of these active sites." Using density functional calculations, Hall's group confirmed, for example, that the catalytic cycle at the iron-iron core of the active site of iron-only hydrogenases involves the oxidation state sequence Fe(II)-Fe(II) ^ Fe(II)-Fe(I)^Fe(I)-Fe(I) rather than the sequence Fe(III)-Fe(III)^Fe(III)-Fe(II) ** Fe(II)-Fe(II) that had been suggested in earlier studies. "Fe(I) is a surprisingly low oxidation state for a biological system," Hall says. He points out that the heterolytic cleavage of dihydrogen is a critical step in the catalytic cycle. The cleavage path in iron-only hydrogenases is thought to involve transfer of a proton to the bridge between the two sulfur atoms in the central core of the active site. However, the composition of the S-S bridge has not been unambiguously defined. Possibilities that have been considered for this bridge include 1,3-propanedithiolate and 2-aza-l,3-propanedithiolate. Density functional calculations by Hall's group reveal that a bridging ligand containing a nitrogen atom provides a kinetically and thermodynamically favorable route for dihydrogen cleavage \J. Am. Chem. Soc, 123, 3828(2001)}. HTTP://PUBS.ACS.ORG/CEN
"Our calculations are the first to confirm suggestions that having 2-aza-l,3propanedithiolate as the bridging ligand in the iron-only enzyme, rather than 1,3propanedithiolate, provides a low-energy pathforthe cleavage andformationof hydrogen," Hall says.
"The process proceeds via reduction of an iron hydride, which has been crystallographically characterized," Rauchfuss tells C&EN. He points out that iron-only hydrogenases contain a strange and still ill-defined cofactor. "Our group and the group in
ACTIVE SITE Oxford University Ph.D. student Sophie E. Lamle investigates activity of hydrogenase docked on an electrode.
At the University of Illinois, UrbanaChampaign, a group led by chemistry professor Thomas B. Rauchfuss is looking into the possibility ofusing diiron dithiolate model compounds as catalysts for biomimetic hydrogen evolution. Last year, the group reported that certain diiron carbonyl dithiolates that bear a structural resemblance to the active site of iron-only hydrogenases are efficient and rugged catalysts for reducing protons to H 2 \Jf.Am. Chem. Soc, 123,9476 (2001)}.
Grenoble have proposed that this cofactor is a2-aza-l,3-propanediothiolate," he says. More recently, Rauchfuss and postdoc Hongxiang Li snowed that iron carbonyl sulfides, formaldehyde, and amines condense to give the proposed azadithiolate cofactor under mild conditions \J. Am. Chem. Soc, 124,726 (2002)}. 'This so-called prebiotic route from simple ingredients suggests that the cofactor might have been selected by nature because it is easily assembled," Rauchfuss says.
"Many challenges remain," he continues. "We have no evidence of any dihydrogen-containing intermediates. Our proton reduction catalyst operates at relatively high overpotential. And we cannot yet elicit any participation from our azadithiolate cofactor, which means either that we are missing something important about the mechanism or that the cofactor assignment is incorrect. Of course, weighing on all modeling efforts is the problem that we lack the elaborate accommodations provided by the protein superstructure of the enzyme." Cammack, at King's College London, points out that the features of the hydrogenase active sites that are important for catalysis place demanding constraints on the structure. He suggests, however, that the synthesis of compounds that imitate the structural features and catalytic activities of hydrogenases offers new possibilities for catalysts in the chemical industry "Progress has been made in constructing models that reproduce the structural features of active sites, though in the short time since the structures were discovered, the activity of the enzymes has not been reproduced," he says. Armstrong is cautious about the possible industrial use of hydrogenases or models of their active sites as catalysts for the reversible oxidation of hydrogen. "The hydrogenase story that is emerging has obvious technological implications," he concludes. "I think this remains more a vision than a reality at the present time. However, it is an important vision." •
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