Trace metals, enzymes, and biogeochemical cycles

Background

Once taken up and bound to microorganisms, some trace metals are incorporated in enzymes that catalyze biochemical transformations at key points in the carbon and nitrogen cycles.

    The carbon cycle

Carbonic anhydrase converts inorganic carbon from the form it takes in sea water (bicarbonate) to a form (CO₂) that can be used in photosynthesis. The carbonic anhydrase enzyme usually contains zinc, which is scarce in surface waters of the open ocean. Because carbon acquisition is essential to cell growth, the amount of zinc may limit the rate of primary production and CO₂ fixation: carbon cannot be acquired if organisms lack zinc, or a means of compensating for its absence.

Cebic scientists are studying in detail the variety of means nature has for fixing inorganic carbon, and how primary producing organisms in marine waters respond to environmental changes in the concentration of metals and CO₂.

Trace metals also participate in the carbon cycle during the degradation of organic carbon molecules. Alkanes are forms of hydrocarbon that are especially rich in energy, making them an attractive energy source for both people and microorganisms. Degradation of alkanes by microorganisms may prove to be an important natural source of atmospheric CO₂, just as alkane degradation by humans (e.g., in car engines and power plants) is an important anthropogenic source. Furthermore, alkane biodegradation removes alkanes from the environment naturally; "seeding" oil-contaminated water with iron may help nature clean up oil spills. Adding naturally occurring elements like iron to sea water, to enhance native microbe populations, is preferable to adding non-native microbes or chemical detergents.

Not much is known about alkane degradation. In the best-studied system, the bacterium Pseudomonas oleovorans, we know that the first step in alkane degradation is catalyzed by an iron-containing enzyme, AlkB. But the mechanism AlkB uses is not well understood. Nor is it well known how widespread the AlkB gene is or whether there are a multitude of other genes and other mechanisms available in nature to degrade alkanes. Studies show that alkanes in the environment degrade completely via natural processes, indicating that alkane-degrading organisms are abundant in nature. Furthermore, degradation occurs in both oxygen-free and oxygen-rich environments, demonstrating that nature has more than one way of degrading alkanes. Cebic aims to survey alkane-degrading organisms, enzymes, and mechanisms in order to learn how alkane biodegradation works, and to determine which mechanisms and enzymes are most important in nature.

    The nitrogen cycle

Nitrogen is abundant, but its most common form—N₂ gas—is too inert to be used directly by most organisms. Nitrogen has to be "fixed"—bound to hydrogen or oxygen to form more reactive compounds—before it can be incorporated into living cells. Once fixed, nitrogen compounds are transformed from one chemical form ("oxidation state") to another as various microorganisms use them to fulfill their energy and cell-building needs. Eventually, fixed nitrogen compounds are converted back to dinitrogen gas.

In oceans, the work of fixing nitrogen is done by nitrogenase enzymes inside a particular genus of photosynthetic cyanobacteria (blue-green algae), Trichodesmium. Trichodesmium's nitrogenase enzyme probably contains both

Trichodesmium

molybdenum and iron. Because iron is scarce in oceans, especially near the surface where Trichodesmium does its work, iron may be the limiting factor in marine nitrogen fixation. Cebic wants to learn how changes in environmental concentrations of molybdenum and iron affect the rate of nitrogen fixation.

Once fixed, nitrogen-containing compounds are acquired by microalgae to be used in cell-building. This is a process of great biogeochemical significance, since the acquisition rate of fixed nitrogen compounds by autotrophs is thought to limit the rate of carbon fixation in the world's oceans—hence the rate of conversion of the greenhouse gas CO₂ to organic carbon forms.

The acquisition of nitrate by microalgae is made possible by a group of enzymes called assimilatory reductases, which come in both nitrite- and nitrate-reducing varieties. Both varieties use iron. Cebic's goal is to determine the relationship between the availability of iron and the activity of nitrate and nitrite reductase enzymes.

Another process occurring in certain bacteria converts fixed nitrogen back to N₂ gas as a side-effect of anaerobic respiration. This process—denitrification via dissimilatory reduction—is driven by dissimilatory reductase enzymes that contain molybdenum, copper, and iron. Cebic seeks a detailed understanding of the process of dissimilatory reduction: how it works, how varied the enzymes are, and how the various reductase enzymes respond to changes in environmental metal concentrations.

Progress

Todd Lane and François Morel of Princeton have identified and characterized a diatom carbonic anhydrase enzyme that can substitute cobalt for zinc without a loss of enzyme activity.

In these experiments, zinc was limited in the diatom growth medium and cobalt was made available. Carbonic anhydrase activity was still observed in the Thalassiosira weissflogii culture in the absence of zinc. Protein purification yielded a cobalt-containing carbonic anhydrase enzyme, which co-chromatographed with the Zn-containing enzyme on an electrophoresis gel, indicating that the new enzyme is just like the old one, except for the metal. EXAFS studies allowed comparison of the coordination environment of the two enzymes, and provided further evidence that the zinc- and cobalt-containing enzymes are otherwise identical. This work was published in Plant Physiology:

Lane, Todd W. and Morel, François M.M. Regulation of Carbonic Anhydrase Expression by Zinc, Cobalt, and Carbon Dioxide in the Marine Diatom Thalassiosira weissflogii. Plant Physiol. 123: 345-352 (2000).

Lane and Morel also identified another carbonic anhydrase enzyme in the same diatom—Thalassiorsira weissflogii—this one containing cadmium. This carbonic anhydrase enzyme, which is different from the zinc/cobalt version, is the first reported case in all of nature of an enzyme that uses cadmium.

This discovery also helps to explain another mystery. It has been known for twenty years that cadmium, like other trace metals, tends to be present in ocean exactly in proportion to other essential nutrients like phosphate, nitrate, and silicate. This would be easy to understand if cadmium had a biological role: essential metals are scarce where nutrients are scarce because the metals are taken in and used by organisms to help them utilize the available nutrients. They are abundant where nutrients are abundant because organisms decay, liberating both nutrients and metals. But why should cadmium, which had no known biological function, behave the same way? Now that we know that cadmium has a biological function, its apparent regulation by marine microorganisms seems less mysterious. This result was reported in the Proceedings of the National Academy of Sciences:

Lane, Todd W., and Morel, François M. M. A biological function for cadmium in marine diatoms. Proceedings of the National Academy of Sciences 97: 4627-4631 (2000).

In a letter to Nature, Jay Cullen and Robert Sherrell of Rutgers, together with Todd Lane and Francois Morel of Princeton, reported more experiments on the cadmium - nutrient connection. They found that the cadmium concentration in phytoplankton collected off the coast of California varies strongly and inversely with concentrations of zinc and CO₂: more zinc and CO₂ means less cadmium, and vice versa. This is precisely what one would expect from a metal used in carbonic anhydrase.

Cullen and the other scientists also observed, in laboratory cultures of T. weissflogii, an inverse correlation between the concentration of the cadmium-containing carbonic anhydrase and dissolved CO₂ and zinc. Taken together, these results—one from the lab and one from the field—strongly support the hypothesis that phytoplankton use cadmium to make carbonic anhydrase, and that this is the reason cadmium is present in sea water in direct proportion to nutrients like phosphorous.

The authors measured these effects (the correlations between cadmium concentration and the concentrations of CO₂ and zinc) as a function of phytoplankton size. They found that the correlation exists in every size class, providing strong evidence that the cadmium-containing carbonic anhydrase occurs in many species of phytoplankton, not just in T. Weissflogii where it was first observed.

One reason this result is important is that it complicates the interpretation of experiments measuring past ocean-nutrient distributions: we now know that the amount of cadmium depends not only on the amount of nutrient, but also on the amount of dissolved CO₂. So cadmium concentrations are not a simple, direct indicator of past nutrient distributions.

Cullen, Jay T., Todd W. Lane, Francois M. M. Morel, and Robert M. Sherrell. Modulation of cadmium uptake in photoplankton by seawater CO₂ concentration. Nature 402: 165-167 (1999).

Carbonic anhydrase is needed in marine bacteria because the C3 photosynthetic pathway, long thought to be dominant in marine microalgae, requires CO₂, while most of the inorganic carbon in oceans is in the form HCO₃^-. But it has long been known that there is another photosynthetic pathway in terrestrial plants, the "C4" pathway, that can start directly from HCO₃^-. It has been suggested, but until now never confirmed, that a C4 pathway exists in marine diatoms. Such a pathway would explain, in part, why photosynthesis in marine diatoms doesn't depend very strongly on the concentration of CO₂.

In a recent Nature letter CEBIC scientists Reinfelder, Kraplei, and Morel reported the discovery of a C4 pathway in T. weissflogii. Reinfelder, et al. followed the radiolabel ¹⁴C from inorganic carbon through to the expected C4-pathway product, malate. They found that when Zn²⁺ levels are low, activity of the dominant enzyme in the C4 pathway, phosphoenolpyruvate carboxylase (PEPcase), is high—evidence that the C4 pathway can compensate for the scarcity of carbonic ahnydrase.

John R. Reinfelder, Anne M.L. Kraeplei, and F.M.M. Morel. Unicellular C₄ photosynthesis in a marine diatom, Nature 407: 996-999 (2000).

Tortell, et al. studied the the affect on carbon acquisition rates on CO₂ levels in Pacific marine phytoplankton. They found that phytoplankton growth rates do not depend significantly on the amount of dissolved inorganic carbon (carbon dioxide and bicarbonate), except at very low levels—surprising, considering that dissolved inorganic carbon is the sole carbon source for marine phytoplankton.

The phytoplankton manage this, the authors argue, by concentrating carbon internally: they take in more carbon than they need (except at the lowest inorganic carbon levels) and maintain a reservoir of carbon. Phytoplankton adapt to low inorganic carbon levels by producing more Rubisco (the enzyme responsible for photosynthesis) and (as noted in the Nature letter by Cullen, et al.) more carbonic anhydrase.

Tortell, Philippe D., Rau, Greg H., and Morel, François M.M. Inorganic carbon acquisition in coastal Pacific phytophankton communities. Limnol. Oceanogr. 45(7): 1485-1500 (2000).

In a letter published in the Journal of the American Chemical Society, chemists Rachel Austin and John Groves, working with microbiologists Huang-Kuang Chang and Gerben Zylstra, demonstrated the efficacy of a new approach to studying alkane-transformation mechanisms: utilizing radical clocks to elucidate enzyme activity in microorganisms.

Radical clocks are compounds that, when metabolized in an enzyme-mediated reaction, report back

GC retention-time trace of metabolism products of norcarane from the AlkB hydroxylase enzyme in E. coli. The first peak indicates the radical product.

information on the mechanism of metabolism. Austin, et al., showed that AlkB - in either a native host (Pseudomonas oleovorans) or a non-native host (E. coli) - metabolizes the radical clock norcarane. When norcarane was fed to both native and non-native hosts of AlkB, Austin and colleagues observed a distribution of "ring-open" and "ring-closed" reaction products. The presence of the ring-opened product indicates a radical reaction mechanism. The ratio of ring-opened to ring-closed products allowed Austin, et al. to estimate the lifetime of the radical reacting with AlkB to be on the order of one nanosecond.

The work of Austin, et al. is the starting point for the exploration of the full range of alkane-degrading mechanisms and organisms in the environment. AlkB is the model system: once the AlkB mechanism is understood in detail, it will be possible to recognize its signature when it occurs in nature, and to detect new alkane-degrading mechanisms (and enzymes) when they appear. These new mechanisms can then be studied using similar techniques, and in time a detailed understanding can be acquired of alkane degradation on a global scale. The work of Austin and colleagues was communicated to the Journal of the American Chemical Society:

Rachel N. Austin, Huang-Kuang Chang, Gerben J. Zylstra, and John T. Groves, The non-heme diiron alkane monooxygenase of Pseudomonas oleovorans (AlkB) hydroxylates via a substrate radical intermediate, Journal of the American Chemical Society 122(47): 11747-11748 (2000).

Ask most scientists about hydrocarbon degradation and they are likely to describe a reaction between an alkane and molecular oxygen. Yet for the last 45 years the scientific literature has included occasional hints of a biological capacity for anaerobic (oxygen-free) alkane degradation. Only in the last few years have pure cultures been isolated and characterized of organisms that can degrade alkanes under anaerobic conditions, using sulfate or nitrate as the terminal electron acceptor.

So and Young have isolated and identified one of the strains of anaerobic alkane degrading bacteria known as AK-01. In this paper, they do some experiments to determine the mechanism of anaerobic alkane degradation by AK-01 and find evidence that the mechanism involves initial addition of a carbon atom at a subterminal position. Work with ¹³C-labeled bicarbonate, which was not incorporated into the alkane transformation product, led them to include that the additional carbon inserted did not come from bicarbonate. This is another step in piecing together a global picture of alkane degradation. Published in Applied and Environmental Microbiology:

C.M. So and L.Y Young, Intial reactions in anaerobic alkane degradation by a sulfate reducer, strain AK-01, Applied Environmental Microbiology 65, 5532-5540 (1999)

    phthalate metabolism

Not all processes related to the carbon cycle involve naturally occurring compounds. Biodegradation by microorganisms is one of the most important pathways for the elimination of toxic human-made organic compounds from the environment. Some microorganisms have developed the ability to utilize toxic (to other organisms) substances as food; in the process they metabolize the toxic organic-carbon compounds to simpler organic carbon compounds that can be degraded further by other organisms. In effect, they return the carbon in these human-made compounds to the natural carbon cycle.

One example is phthlates, plasticizers used in manufacturing soft polyvinyl-chloride. Phthalates are ubiquitous trace pollutants in soils amended by sludge from wastewater treatment plants, and in marine environments near wastewater-plant outflows. Certain organisms, like Burkholderia cepacia ATCC 17616, can metabolize phthalates.

In this work, already mentioned in the section on chelation, uptake, and binding, Cebic scientists Hung-Kuang Chang and Gerben Zylstra of Rutgers University identified a pathway for phthalate uptake in B. cepacia by expressing in E. coli the B. Cepacia gene (OphD) thought to be responsible for phthalate uptake. E. coli does not naturally metabolize phthalate, and it lacks the ability to transport phthalate into its cells. But the mutated E. coli was able to efficiently take up phthalate, showing that OphD is, indeed, responsible for creating the phthalate uptake machinery. However, Chang and Zylstra also found that "knocking out" the OphD gene from B. cepacia failed to inhibit phthalate uptake, which suggests that the organism must have an alternative phthalate-uptake pathway. This work was published in the Journal of Bacteriology:

Chang, H.-K., and Zylstra, G. J. Characterization of the phthalate permease OphD from Burkholderia cepacia ATCC 17616. J. Bacteriol. 181: 6197-6199 (1999).

    dissimilatory nitrite reduction

Ammonia-oxidizing lithoautotrophic ("nitrifying") bacteria are unusual. They are neither heterotrophic like animals (using organic carbon molecules as a source of carbon and energy, and oxygen gas as a terminal electron acceptor) or photoautotrophic like plants (using CO₂ as a carbon source and the sun as an energy source). Like heterotrophic organisms, these bacteria get energy from a chemical (in this case an inorganic chemical: ammonia); like photoautotrophic organisms they get the carbon they need from CO₂.

Ammonia-eating bacteria are also peculiar in that they play two distinct and inverse roles in the nitrogen cycle: they act as both nitrifiers and denitrifiers.

"Classical" denitrifying bacteria are anaerobic; they use nitrites and nitrates in place of oxygen as terminal electron acceptors during anaerobic respiration. In the process nitrites are transformed into nitrogen-oxide gases, NO and N₂O, a process called denitrification via dissimilatory reduction.

Nitrosomonas marina and other ammonia-oxidizers use nitrites as terminal electron acceptors, too, when oxygen levels are low. But they can also use oxygen as a terminal electron acceptor. And oxygen is essential in another process: the extraction of energy via the conversion of ammonia to hydroxylamine, an example of a process known as nitrification. These curious bugs act like anaerobes but require oxygen, and they nitrify and denitrify at the same time.

In a paper published in Applied and Environmental Microbiology, Karen Casciotti and Bess Ward of Princeton searched several varieties of ammonia-oxidizing bacteria for the gene nirK, which codes for a copper-containing dissimilatory nitrite-reductase enzyme in classical denitrifying bacteria. Their goal was to determine whether the NirK enzyme was responsible for denitrification in ammonia-oxidizing bacteria.

The situation, they discovered, is complex. NirK was found in some ammonia-oxidizers, but not in others. In those organisms where it was detected, substantial variation in the nirK genetic sequence was observed. The organisms found to contain nirK fell into two main groups, divided according to similarities in the nirK sequence. The first group contained most of the nirK-containing ammonia-oxidizing bacteria. The second group contained mainly classical anaerobic denitrifying bacteria, but it also contained one ammonia-oxidizing bacterium, collected from the Chesapeake Bay.

The nirK gene was not detected in several other ammonia-oxidizing bacteria, suggesting an even greater variety of dissimilatory reductase enzymes. These bacteria may have contained genes resembling nirK, but they were not similar enough to yield a PCR match.

The nucleic-acid sequences of all the organisms in both nirK-containing groups were similar in the region that codes for the copper-containing active site. This means that the resulting enzymes are probably functionally quite similar, even if they vary considerably in other parts of the genetic sequence.

Casciotti, Karen L. and Ward, Bess B. Dissimilatory nitrite reductase genes from autotrophic ammonia-oxidizing bacteria. Appl. and Env. Microbiology .

The most inert hydrocarbon is methane (CH₄). In this report, CEBIC scientists Groves, Austin, and Tarr team up with Brazeau and Lipscomb from the University of Minnesota to investigate the reaction mechanism of one of the two metalloenzymes that can hydroxylate methane. Using the substrate probe norcarane, the authors detected evidence for both a radical and a cationic intermediate in its metabolism. Furthermore, the authors show that the high-valent intermediate known as compound Q is the chemical form of the active site that reacts directly with the alkane. The presence of 3-hydroxymethylcyclohexene among the products is clear evidence for homolytic cleavage of the C-H bond, consistent with an oxygen rebound mechanism. The authors hypothesize that the cationic product results from a subsequent electron transfer to a hydroxo-Fe(III)Fe(IV) enzyme species termed R before O atom rebound can occur. The estimate of the norcarane radical lifetime with sMMO ranges from 0.02-0.1 ns, significantly shorter than the lifetime detected by Groves, Austin, Zylstra, and Chang with the histidine rich diiron metalloenzyme AlkB, but still long enough to be considered a distinct intermediate.

B.J. Brazeau, R.N. Austin, C. Tarr, J.T. Groves, and J.D. Lipscomb. Intermediate Q from soluble methane monooxygenase Hydroxylates with the Mechanistic Substrate Probe Norcarane: Evidence for a Stepwise Reaction, J. Am. Chem. Soc. 123: 11831-11837 (2001).

X-ray absorption spectroscopy centered on the core electrons in Zinc indicates that the Zinc atom at the active site of the carbonic anhydrase from this diatom is very similar to the active site of the mammalian alpha-carbonic anhydrase enzyme and the gamma carbonic anhydrase from methanoarcheon, all of which have three histidines and one water molecule. This active site is different from the active site found in the beta-carbonic anhydrases from plants, which contain a zinc atom coordinated to two cysteines and one histidine and one water molecule. Since there is little sequence homology between the alpha, beta, gamma, and diatom carbonic anhydrase sequences, the authors hypothesize that the structural similarities in the active sites of the alpha, gamma and T. weissflogii active sites represent a case of convergent evolution.

E.H. Cox, G.L. McClendon, F.M.M. Morel, T.W. Lane, R.C. Prince, I.J. Pickering, and G.N. George. The active site structure of Thalassiosira weissflogii Carbonic Anhydrase I, Biochemistry 39: 12128-12130 (2000).

What is the physiological role for nitrite reduction in organisms that oxidize ammonia for fuel? The answer is not yet known, although the recent work of Casciotti and Ward indicates that copper-containing dissimilatory nitrite reductase genes with high sequence homology to the classic heterotrophic denitrifiers are found in several ammonia-oxidizing bacteria isolated from marine environments. The authors developed a new set of primers for the detection and amplification of nitrite reductase genes that may enable detection of nitrite reductase genes with greater sequence diversity than previously developed primers. Application of the primers developed by the authors may facilitate better surveys to determine the extent to which ammonia-oxidizing bacteria contain nitrite reductase genes. The present work does not identify whether the genes are expressing a functional nitrite reductase enzyme, although the authors point to the highly conserved copper-binding region as suggestive that the gene product remains functional.

K.L. Casciotti and B.B. Ward. Dissimilatory Nitrite Reductase Genes from Autotrophic Ammonia-Oxidizing Bacteria, Applied and Environmental Microbiology, 2213-2221 (2001).

Lane and Morel found that at low concentrations of CO₂ (100 microatmospheres), growth of T. weissflogii is diminished when Zinc is limited to 3 picomolar in comparison to cultures grown at 15 picomolar. If the CO₂ concentration is increased to 750 mictoatmospheres, the effect of limiting zinc is partially alleviated. Addition or 21 picomolar inorganic cobalt to the zinc limited cultures completed removed the growth limitations imposed by the low zinc concentrations. The authors also assessed carbonic anhydrase activity and found that the activity levels closely mirrored the levels of protein determined by a western blot analysis, except at high CO₂ levels where the western blot analysis appears to under-represent the amount of active CA in the cells. Significantly, the carbonic anhydrase activity was the same for cells grown in zinc-replete or cobalt-replete environments. If cells were taken from a high CO₂ environment and placed in a low CO₂ environment in either zinc- or cobalt replete media, carbonic anhydrase levels were seen to rise after about 5 hours and reach steady state levels after about 18 hours. Carbonic anhydrase levels were higher in the presence of light than in its absence, again consistent with carbonic anhydrase playing a role in delivering CO₂ to Rubisco for carbon fixation. Together, these experiments indicate that T. weissflogii has a CA-dependent mechanism for carbon concentration and that the carbonic anhydrase from this marine diatom can effectively use cobalt in place of zinc. Cox et al. have determined the active site structure of this enzyme (see paper 6) using EXAFS. Thus, details of the chemistry of this intriguing metalloenzyme are beginning to emerge.

T.W. Lane and F.M.M. Morel. Regulation of Carbonic Anhydrase Expression by Zinc, Cobalt, and Carbon Dioxide in the Marine Diatom Thalassiosira weissfloggi Plant Physiology 123: 345-354 (2000).

Cyanobacteria from the genus Trichodesmium are key players in nitrogen fixation in the marine environment. They are also photosynthetic organisms, which presents an apparent paradox as molecular oxygen is the byproduct of photosynthesis (from the oxidation of water) and is toxic to the metalloenzyme nitrogenase, which fixes nitrogen. In this report, the authors unravel this mystery, identifying that Trichodesmium both spatially and temporaly separate photosynthesis and nitrogen fixation. Using fast repetition rate fluoremetry to monitor chromophores and following oxygen production and carbon and nitrogen fixation, the authors found that photosynthetic carbon fixation increased in the morning and declined at midday, which is when nitrogen fixation activity peaked. Nitrogen fixation rates remained high for about six hours in the middle of the day. After nitrogen fixation declined, photosynthetic carbon uptake increased again. This observation was made both in the field and in the laboratory where Trichodesmium cultures were studied. The authors observed that the rate of oxidation of the primary electron acceptor in photosystem II declines during the day. If they blocked the linear electron transport on the acceptor side of PSII (either forcing all the components in the plastoquinone pool to be reduced or oxidized) they saw an immediate decline in nitrogenase activity in aerobic cultures. Anaerobic cultures showed nitrogenase inhibition only when both photosynthetic and respiratory pathways were shut down but not when quinone A oxidation was inhibited. The authors conclude from this data that nitrogenase is protected from oxygen by electrons supplied by PSII, which facilitate the scavenging of molecular oxygen by PSI.

The following picture emerges. Light stimulates photosynthesis, which poises the plastoquinone pool in the reduced state and provides energy and reducing agents for carbohydrate synthesis. Respiration provides the carbon skeletons needed for amino acid synthesis (completing the synthesis requires fixed nitrogen) and further reduces the plastoquinone pool. The reduced pool creates a negative feedback that leads to the down-regulation of PSII. PSI remains active, receiving electrons that are used to reduce oxygen to hydrogen peroxide. When PSII is down-regulated, oxygen consumption exceeds oxygen production and nitrogenase activity can be established. As carbohydrates are consumed, respiratory electron flow to PSI declines and oxygen levels begin to rise. Nitrogenase activity does not return until the following day, when the cycle repeats.

I. Berman-Frank, P. Lungren, Y.-B. Chen, H. Küpper, Z. Kolber, B. Bergman, P. Falkowski. Segregation of Nitrogen Fixation and Oxygenic Photosynthesis in the Marine Cyanobacterium Trichodesmium, Science 294: 1534-1537 (2001).

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