Scholar Profile

Michael P. Hendrich

Professor
Department of Chemistry
Carnegie Mellon University
4400 Fifth Avenue
Pittsburgh, PA 15213
Voice: 412-268-1058
Fax: 412-268-1061
Email: hendrich@andrew.cmu.edu
Personal Homepage
1995 Searle Scholar

Research Interests

The Characterization of a New Metal Cluster in Enzymes.

Plants receive nitrogen for growth from fertilizers via a pathway dependent on soil bacteria which feed on ammonia. In the soil bacteria nitrosomonas, at least two enzymes are required, ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO), which sequentially oxidize ammonia to hydroxylamine, and then to nitrite. New advances in EPR spectroscopy have provided signatures from a novel type of an enzymatic metal cluster in the protein HAO. This large molecular weight protein is a trimer, with each monomeric polypeptide unit contains eight heme groups. Oxidation of the substrate hydroxylamine occurs at a covalently-linked diheme cluster; we believe this is the first known occurrence of this type of cluster in biology.1 Metal clusters, of which only approximately a dozen are known, perform unique and pivotal functions in biological processes. Thus, the discovery of a new cluster may impact other biological domains.

Recently, we have completed experiments to verify the existence of this cluster. In an electrochemical titration study, both EPR and optical spectra of the eight hemes groups of HAO were monitored. The enzymatic active site of HAO is known to contain one heme with midpoint potential of -322 mV. We have determined, however, that the characteristic EPR signal of this active site vanishes at a much higher potential of -150 mV. Moreover, this potential coincides with the midpoint potential of another heme in HAO. This result provides further evidence in support of the diheme cluster, by ruling out the possible existence of an active site composed of a protein radical coupled to a single heme.

The mechanism of the enzymatic cycle of HAO is not known, but we believe that a diheme cluster would facilitate the 4-electron oxidation of hydroxylamine in a two-step process. Studies are in progress to observe intermediates in this mechanism. Another protein from nitrosomonas, cytochrome c554, accepts electrons from HAO for eventual delivery to cytochrome oxidase. However, this protein has been suspected of having an enzymatic function in which an intermediate in the hydroxylamine oxidation is a substrate. We have discovered new EPR signals from cytochrome c554 in support of an enzymatic role.

An important advance in EPR spectroscopy has facilitated these discoveries: the ability to quantitatively study paramagnetic centers possessing integer electronic spin. Further instrumentation advances are to be completed this year which raise the microwave frequency of the measurements. This increase in frequency enables investigations of metalloproteins that would otherwise be impossible.