CHAPTER 1: Introduction to the Biological Chemistry of Nickel

Nickel was first identified in the 1960s as an essential nutrient for strains of Hydrogenomonas that were growing chemolithotrophically on the simple gasses H₂, O₂, and CO₂.¹  This discovery was followed by the observation that Methanobacterium thermoautotrophicum also required nickel supplementation of the growth media, but only if the media had not had any contact with stainless steel equipment, suggesting that even the trace nickel leaching from transient contact with syringe needles was sufficient for healthy growth.²  A clear function for nickel was established with the finding that nickel is an essential component of Jack Bean urease,³  which explained the nickel dependence of soybeans grown with urea as the nitrogen source.⁴  Subsequently, it was found that nickel is required for the production of carbon monoxide dehydrogenase in Clostridium pasteurianum.⁵  It is now well established that nickel is an essential cofactor for a variety of enzymes, many of which have been described in earlier reviews (e.g., see ref. 6–10).

On a global scale, the importance of nickel in biology is highlighted (Figure 1.1) by the discussion in Chapter 2 about the influence of nickel-utilizing organisms on the earth’s geochemistry, and vice versa. It is striking that many nickel enzymes consume or produce small molecule gasses, they are often required for anaerobic microbial metabolism, and they contribute to the global elemental cycles.^11–13  These types of chemistry highlight the ancient history of nickel as a biological cofactor for organisms that evolved early on, during the origins of life as we know it.¹⁴  In fact, given that nickel-enzyme metallocenters are often complex, multi-metal clusters, it has been suggested that these metal complexes may mimic the minerals that catalyzed the first abiotic reactions where life emerged.^15,16  The link between the evolution of nickel use and nickel availability is exemplified by the presence of nickel superoxide dismutase (NiSOD) in many marine organisms (Chapters 2 and 9). SODs provide critical protection from reactive oxygen molecules that are byproducts of living in oxygen-containing environments, and there are three distinct versions of this enzyme that catalyze the same reaction but that contain different types of metal.¹⁷  It is likely that NiSOD was favored in some types of settings, such as marine environments, over other metal-utilizing SODs because nickel was more accessible than other types of metals, but that development may in turn have resulted in nickel depletion of those same environments (Chapter 2).

As of yet, a required bioactivity for nickel has not been identified in human cells. However, nickel is ingested in many types of food,^18,19  and nickel deprivation has a negative impact on various physiological factors in animals.^20,21  Nickel circulates bound to serum albumin, as well as in trace amounts to several types of amino acids and α2-macroglobulin (nickeloplasmin),²²  and is widely distributed in tissues throughout the body.²¹ 

Human exposure to nickel is also impacted by anthropogenic activities, such as metal-related industries and industrial pollution.¹⁹  This exposure can lead to toxicity, an occupational hazard of industries such as nickel mining and refining or stainless steel manufacturing and manipulation, with nickel being a well-established carcinogen.¹⁹  Chapter 3 provides an extensive discussion about the significant epigenetic consequences of nickel exposure in humans, such as changes to DNA methylation patterns and to levels of microRNAs, both of which promote carcinogenesis. Nickel is also an allergen, and everyday exposure to nickel in common household items, such as cooking utensils, jewelry, and money, can cause allergic reactions.²³  For example, in the early 2000s the Euro coins elicited strong skin reactions in people with nickel allergies, possibly due to solubilization of the metal by human sweat.^24,25  Later on, legislation was introduced to limit the amount of nickel contained in manufactured items in an effort to reduce human skin contact with nickel.²⁶  The mechanisms of allergic reactions to nickel are multifaceted,^25,26  but activation of the human Toll-like receptor 4 has a key role in the inflammatory response to nickel.²⁷  Furthermore, modeling and mutagenesis of several non-conserved histidines revealed the location of a putative nickel ion binding site on TL4, and resolved the long standing mystery behind different nickel sensitization in humans versus in mice. These results also raise the questions of whether there is some type of selection pressure for the distinct sequence motif in human TL4, and why all humans are not sensitized to nickel.

Multiple nickel enzymes are found in Nature. Consideration of how nickel ions interact with the enzyme proteins, as well as the proteins that support nickel homeostasis, has revealed some common themes about these complexes. Chapter 4 surveys our knowledge about the coordination chemistry of nickel in biology, which has been greatly augmented by the study of smaller peptide nickel complexes. Some binding preferences can be extracted, and these preferences are emphasized in proteins dedicated to nickel storage and distribution. However, it is clear that nickel is not inordinately exclusive and can be comfortably accommodated by a variety of protein sites. Furthermore, as shown below, nickel catalyzes a wide range of chemical reactions when bound to enzyme proteins. Our current understanding about many of these nickel enzymes is discussed in detail in Chapters 5–11 of this book, along with outstanding questions.

As mentioned above, the first enzyme that was recognized to use nickel as a cofactor was urease, which catalyzes the hydrolysis of urea.³  This enzyme is a key virulence factor in multiple pathogenic bacteria, as highlighted in Chapter 16. The products of urease provide a source of nitrogen, buffer the local microenvironment, and/or contribute to pathogenesis via the formation of infection stones. Furthermore, as discussed in Chapter 5, urease can also be a significant agricultural problem. Chapter 5 describes the many structural and enzymatic studies performed with urease and various inhibitors, which together form the foundation for a detailed mechanism of action. This knowledge can now be used to guide the design of better inhibitors, and also informs the discussion of why nickel is used in urease, as opposed to more commonly available metal ions that, at first glance, appear suitable for catalysis of this chemical reaction.

Another nickel enzyme is [NiFe]-hydrogenase, which catalyzes the relatively simple reversible reaction between two protons and two electrons to generate hydrogen gas; the direction of the reaction depends on the biochemical context of individual enzymes.^28,29  However, Chapter 6, which focuses on the results from X-ray crystallography, describes a complex system that involves multiple enzyme states. Furthermore, several mechanisms by which [NiFe(Se)]-hydrogenase enzymes prevent or deal with inactivation by poisons such as oxygen have been uncovered. This enzyme is also highlighted in Chapter 16 as a virulence factor for several human pathogens.

Chapter 7 discusses the extensive structural and biochemical studies that are unveiling what happens at the metal clusters of carbon monoxide dehydrogenase and acetyl coenzyme A synthase.³⁰  These enzymes catalyze one-carbon chemistry that is a part of the basic metabolism of many organisms, serving as the entry or exit point of CO₂ and/or CO. This chapter also highlights the critical functions provided by the extensive protein scaffolds, which facilitate the handling of the gaseous and potentially toxic small molecules involved. The ramifications of the possible applications of this chemistry, either through harnessing the enzymology or mimicking Nature’s strategies, in terms of generating solutions to global climate change and sources of sustainable energy are considerable.

The central role of nickel in the global carbon cycle is also emphasized in Chapter 8, which describes methyl coenzyme M reductase (MCR).¹²  This enzyme catalyzes the final step for methane production in methanogenic archaea, which are pervasive in many anaerobic environmental niches around this planet, and it can also catalyze the reverse reaction in methanotrophs. The cofactor in MCR is an unusual nickel-loaded tetrapyrrole, called F430 because of the strong absorption band of the isolated (and inactive) Ni(ii) form. As discussed in Chapter 8, the elucidation of the detailed mechanism of action of MCR is now underway via the use of rigorous anaerobic techniques, and a combination of spectroscopy, enzymology, and computational analysis.

Chapter 9 examines NiSOD, which is one of several SODs that each uses a different metal to catalyze the disproportion of toxic superoxide, a key reaction in the self-protection mechanisms of organisms with an aerobic lifestyle.¹⁷  Although the structure of NiSOD is quite distinct from those of the other metal SODs, it has converged to meet the same chemical requirements while adapting the particular chemistry of nickel. In addition, because the complete nickel-containing active site is contained within a short sequence at the N-terminus of the protein, extensive studies of peptide models in combination with those of the protein have provided in-depth information about the mechanism of action of this enzyme.

Although we know about multiple uses for nickel in biology, it is likely that there are more nickel systems to be found. The latest nickel enzyme to be added to the list, lactate racemase (Chapter 11), was only characterized a few years ago.³¹  An operon encoding the lactate racemase activity, larA–E, inducible by l-lactate, was identified in Lactobacillus plantarum,³²  but the activity could not be transferred upon expression of this operon in a heterologous host. In this case, knowledge about nickel homeostasis systems came into play, because a second operon was identified just upstream of larA–E, which includes genes that resemble those encoding nickel uptake transporters found in many other microorganisms.^33,34  This clue led to the demonstration that the lactate racemase protein, LarA, uses a nickel ion for activity, and that the enzyme requires a few of the other Lar proteins to produce active enzyme.³¹  Subsequent mass spectrometry coupled with crystallography revealed an unprecedented nickel complex that includes carbon and sulfur coordination via a derivatized nicotinic acid mononucleotide.³⁵  Lactate racemase and the unusual nickel cofactor is described in Chapter 11, along with the pathway for the assembly of this complicated cofactor by the accessory proteins.³⁶  Future analysis will not only reveal the mechanism of action of this new and metabolically expensive cofactor, but also how widespread it is in biology.

It is not always apparent why nickel was selected as a cofactor for certain enzymes, but it is clear that once this selection was made the active sites of nickel enzymes must be loaded with nickel, and not with other metals, to catalyze the designated activity. The obligations of metal selectivity in a nickel enzyme are highlighted by the case of glyoxalase I (Chapter 10). This enzyme, which contributes to the detoxification of methylglyoxal and other α-ketoaldehydes, is a zinc enzyme in some organisms and is nickel-activated in others. Crystal structures of members of the two classes of enzymes reveal a surprising three-dimensional similarity. Chapter 10 discusses the careful and detailed studies that are uncovering the subtle features that dictate the selective metal-activation profiles of these enzymes.

Another example of the impact of correct metal occupancy is the enzyme acireductone dioxygenase (ARD). Initial studies of the methionine salvage pathway revealed that one of the intermediates in this pathway, an acireductone, can be processed by two enzymatic reactions into different sets of products.³⁷  Further studies of the Klebsiella pneumonia system revealed that the reactions are catalyzed by the same protein loaded with different metals.³⁸  When loaded with iron, the enzyme produces the α-keto acid precursor that can be recycled back to methionine, but when loaded with nickel or cobalt, the enzyme catalyzes an off-pathway reaction that results in the formation of several different products including carbon monoxide. Although the two forms of the enzyme can be separated chromatographically, suggesting structural differences,^39,40  spectroscopy and mutagenesis indicate that the two active sites are very similar,^41,42  so it must be that the distinct chemistry is generated by subtle changes. The mouse ARD also has a similar metal-dependent switch in activity,⁴³  and given that the mammalian enzyme has been implicated in various disease states, metal replacement at the active site of ARD raises the possibility of another route for nickel toxicity in humans.

Nickel is near the top of the Irving–Williams series,⁴⁴  and so it has a higher inherent affinity for protein metal-binding sites than metals at the other end of the series, such as manganese and iron. This means that unprotected nickel ions are toxic because they can replace cognate metals, resulting in inactive enzymes or off-pathway reactions. Metal substitution is a potential mechanism of nickel toxicity in humans, as discussed in Chapter 3, since nickel can displace the iron from the active sites of dioxygenase enzymes such as histone demethylases, with ensuing deleterious consequences to the epigenome. However, this problem also has to be dealt with in organisms that employ nickel. For example, Escherichia coli requires nickel as a cofactor of several [NiFe]-hydrogenase enzymes (Chapter 6) expressed under anaerobic metabolism,^28,45  as well as in glyoxylase I (Chapter 10). However, nickel is toxic to this organism.⁴⁶  Although there may be more than one deleterious impact of nickel,⁴⁶  in bacteria fed glucose or fructose as carbon sources, a key target is the glycolytic enzyme fructose-1,6-bisphosphate aldolase, which is disrupted because nickel supplants an allosteric zinc ion.⁴⁷ 

To deal with the toxicity of nickel while ensuring that nutritional requirements are met, organisms that employ this metal ion have multiple support systems to control availability and distribution. One central aspect of nickel homeostasis is the import and export of the metal ion across the membrane(s), which is the responsibility of the transporters that are discussed in detail in Chapter 12. There are multiple types of transporters that vary in mechanism, selectivity, and proficiency of nickel uptake, highlighting the importance of this job and also the need for organisms to adjust to environmental conditions that vary in nickel availability and speciation. The extent of these transporters in microbial genomes underlies the scope of nickel use in biology,^33,48  and the presence of a nickel transporter can also contribute to the identification of new nickel enzymes, as in the case of lactate racemase (Chapter 11).³¹ 

The expression of these nickel transport systems is frequently regulated by nickel-responsive sensors called metalloregulators, which may also control the production of other components of the nickelome (Chapter 13). These regulators are typically cytoplasmic nickel-responsive transcription factor proteins,^49,50  but two-component systems that allow metal sensing in the periplasm (also described in Chapter 15),^51,52  RNA-dependent regulation,⁵³  and indirect pathways³¹  have all been identified. As discussed in Chapter 13, the issue of metal selectivity is critical for these biomolecules. There are now multiple examples of nickel metalloregulators, and detailed studies have revealed that nickel-selective activity is managed by a combination of metal affinities, the allosteric impact of cognate metal binding on DNA binding, as well as metal availability in the different cellular contexts. In addition, several interesting themes are emerging, such as the observation that nickel tends to bind to the regulators of nickel import with different coordination geometry than when bound to the regulators of export.

Ensuring that nickel ions, and not other types of metal ions, are delivered to the nickel enzymes is not a trivial problem. One means of governing the allocation of nickel ions is through the use of metallochaperones, which are proteins that deliver nickel to the nascent nickel enzymes and help to assemble the active site metallocenters. As discussed in Chapter 14, metallochaperones dedicated to the production of [NiFe]-hydrogenase, urease, carbon monoxide dehydrogenase, and lactate racemase have been identified and characterized. Some of these proteins also store nickel and can bind multiple nickel ions in histidine-rich domains much like the his-tags that are often used for affinity chromatography in recombinant protein technology (Chapter 17). However, questions remain about the location and selectivity of metal acquisition by these proteins, as well as the metal delivery mechanisms. Furthermore, it is not clear whether production of the other nickel enzymes also involves a nickel metallochaperone, and, if so, which proteins could serve such roles.

Given that these nickel systems do not function in isolation, but in the complicated context of living organisms, it is perhaps not surprising that there is significant overlap with many other biological pathways. In particular, it is clear that there is often cross-talk with other transition metal systems, which is manifest in different ways. For example, in Streptomyces coelicolor the nickel-responsive transcription factor Nur represses the gene encoding the iron-utilizing SOD in favor of NiSOD, allowing the bacteria to reduce iron utilization under aerobic conditions (Chapter 9).⁵⁴  Similarly, it is clear that there is synergy between nickel and iron regulatory systems in other bacteria such as E. coli and Helicobacter pylori.^55,56  A critical point of overlap is at the membrane, where the uptake and efflux transporters must obtain sufficient nickel in environments that may vary in nickel availability as well as the relative levels of other, potentially interfering, metals. This issue is explored in Chapter 15, which discusses nickel homeostasis in the context of other biologically-relevant transition metal cations. How nickel selectivity can be achieved is exemplified by CnrX, the periplasmic metal-sensor component of a signaling system that regulates the expression of a nickel–cobalt exporter. Structural and biochemical analysis of this protein revealed that nickel and zinc have distinct coordination environments, and that only the ligand set recruited by nickel leads to signal propagation.

Besides being a fundamental aspect of life, the biological chemistry of nickel factors has a variety of possible applications. Given the nickel requirements of some types of pathogenic bacteria (Chapter 16), and the lack of a nutritional use for nickel in humans, these systems are tantalizing targets for the design of new antimicrobial strategies (Chapters 4 and 17). One possible strategy to treat infections by organisms that need nickel could be a diet low in nickel. For example, in a pilot study the treatment of H. pylori infections by standard triple therapy was more successful for patients who refrained from eating foods with high nickel content and using stainless steel utensils.⁵⁷  However, this goal might be difficult to achieve in larger populations because the metal is ubiquitous and there are many variables that impact the nickel content of food and water, so it is not trivial to control or eliminate nickel intake from the human diet.¹⁸ 

A more effective approach may be to target the support systems that accompany the nickel enzymes. Not only do pathogenic organisms use nickel as a cofactor in enzymes such as urease or hydrogenase,^11,58,59  they have multifaceted systems to ensure sufficient nickel accumulation and distribution. For example, Chapter 16 provides a detailed description of some of the unique features of nickel homeostasis in H. pylori, including multiple importers and metallochaperones, which have presumably developed to allow this pathogen to survive in the inhospitable environment of the human stomach. The mechanistic details of how these systems function may provide leads for new antimicrobials.

Finally, the nickel binding properties of the his-tag, a short histidine-rich peptide typically fused to either end of recombinant proteins, have led to one of the most common methods of protein purification through immobilized-metal affinity chromatography (IMAC), as well as various other types of analysis and applications. This nickel-binding sequence is discussed in Chapter 17, which also describes how knowledge of nickel proteins is being developed to detect and/or remove nickel contamination in the environment.

In summary, the nickel systems that we know about play critical roles in biology, they are intricate and diverse, and they exhibit complex biological chemistry that is being revealed through the application of a combination of methods drawn from multiple disciplines. Have we identified all of the functions of nickel in biology? The discovery of a new nickel enzyme just a few years ago (Chapter 11), bearing a completely novel metallocofactor, suggests that there may be other ways that Nature employs nickel. Future experiments may also address the question of whether there is a specific function for nickel in humans, or if the nutritional requirement is instead dictated by our intimate relationship with our microbiome.

Another complicated issue is how the activities of all of the different components of nickel homeostasis are coordinated together within a living organism, and how they accommodate changes in the growth environment or in metabolic requirements. Along the same lines, what types of nickel complexes are available in different ecosystems, and how actively do organisms scavenge for nickel? Is the recent characterization of the metallophore staphylopine,⁶⁰  a nicotianamine-like chelator excreted by Staphylococcus aureus to gather nickel and other metals, a common theme for nickel import? And on the other hand, given the nickel requirements of human pathogens, is controlling nickel availability a component of nutritional immunity as it is for other metals?^61,62  These, and other questions, will be addressed as we continue to explore the fascinating world of the biological chemistry of nickel.

The author would like to thank the Natural Science and Engineering Research Council of Canada (NSERC) as well as the Canadian Institutes of Health Research (CIHR) for funding, as well as members of the Zamble Lab for their insight and suggestions.