Biological inorganic chemistry is a fascinating and diverse discipline which, as the name implies, draws on aspects of both inorganic coordination chemistry and biochemistry. The subject spans an enormous variety of topics, ranging from chemical synthesis and characterization of novel metal complexes for use as models, to bioinorganic enzymology, and to medicinal chemistry. A unifying theme in my own research has always been a keen interest in reaction mechanisms at the molecular level, and I am especially interested in the reactions of metalloenzymes. Currently I am concentrating my efforts on two bacterial enzymes: ammonia monooxygenase (AMO), which oxidizes ammonia to hydroxylamine, and hydroxylamine oxidoredutase (HAO), which subsequently oxidizes the hydroxylamine to nitrite (Eqns. 1, 2).
These reactions are part of the ecologically important process known as "nitrification", which is in turn part of the biological nitrogen cycle.
Remarkably, all of the enzymes from the nitrogen cycle which have been identified to date are in fact metalloenzymes, which utilize a wide variety of transition metals to catalyze, with a high degree of specificity, the transformations between N-containing species. This fact, coupled to the inherent richness of inorganic nitrogen chemistry itself, has made this area a fruitful hunting ground for the bioinorganic chemist. However, for a variety of reasons, the process of nitrification has not been as thoroughly investigated as the other processes in the nitrogen cycle.
An understanding of the two processes of nitrification poses two distinct chemical challenges. AMO, which catalyzes the oxidation of NH3, has not been purified from an autotroph, and is the least studied enzyme in the nitrogen cycle. Indeed, one of the few things known about AMO is that it contains at least one, and more probably multiple Cu centers. Thus, the immediate challenge posed by AMO is one of purifying and characterizing it at the most basic level. Fortunately, a recent breakthrough with two related enzymes suggests a way by which AMO might now be purified, and I plan to investigate this possibility. I then plan to probe the CuII centers of AMO using pulsed-epr techniques, which can provide information about the types of ligands coordinated to the Cu centers, and also information about the arrangement of the Cu atoms with respect to each other.
HAO, which catalyzes the hydroxylamine oxidation, has been much more studied than AMO, and in 1997 its crystal structure was determined. The fascinating feature of HAO is its remarkable complexity. The enzyme appears to be a trimer, with a molecular mass of -67 kDa per monomer. Each monomer contains seven c-type hemes, and a novel eighth heme known as P460, which is the site of hydroxylamine oxidation (Figure 1).
This makes HAO perhaps the most complex heme protein discovered to date. The chemistry of HAO has reached the ideal stage of development for performing detailed mechanistic studies; the molecule is well characterized and its basic reactivity is known, but many important questions remain unanswered. I will call on my knowledge of photochemical and stopped flow techniques to investigate transient processes during reactions of HAO. Also, as with AMO, I will use pulsed EPR techniques to probe the complex structure of HAO.