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Assistant Professor of Biochemistry and Biophysics
Ph.D. Stanford University 2000

Metalloenzyme Reverse-Engineering. The redesign and optimization of enzymes for industrial, environmental, and biomedical applications is an active area of research, as is protein de novo design. However, site-directed mutagenesis and de novo protein design are limited by our poor understanding of protein structure-function correlations. Directed evolution is a dramatically successful approach for the optimization of enzymes towards non-native substrates. However, little effort has been devoted to understand how seemingly random mutations distant from the active site can have such a powerful effect on enzyme activity.

We are combining directed evolution with NMR spectroscopy to study how mutations distant from the active site improve enzyme activity towards non-native substrates. We are targeting two metalloenzymes, tyrosinase and an extradiol dioxygenase, for several reasons. (1) Their small size, simple active sites (binuclear Cu in tyrosinase, mononuclear Fe in extradiol dioxygenase), lack of additional required cofactors or cosubstrates (besides O₂), and well-characterized structure and catalytic cycle make them ideal for detailed studies of active site tuning. (2) These enzymes catalyze chemically difficult and still poorly understood reactions (hydroxylation of an phenol for tyrosinase, oxidative ring cleavage for extradiol dioxygenase), thus rendering rational mutagenesis useless. (3) Both enzymes have been targeted for use in bioremediation of intractable pollutants such as halogenated arenes, but are poor catalysts towards substrates with electron-withdrawing substituents such as Cl-. This provides a clear rationale for enzyme redesign by directed evolution. By generating a series of mutants with improved activities towards poor substrates, we then apply NMR spectroscopy to probe how changes in the protein structure, dynamics, and active site electronic structure lead to altered activities.

15N NMR spectrum showing the paramagnetic shifts of amides that hydrogen bond to a [2Fe-2S] cluster.

Tyrosinase. Tyrosinase is a small, ubiquitous enzyme with a binuclear Cu active site that catalyzes the ortho-hydroxylation of a phenol with incorporation of one atom from O₂ (the other atom becomes water/hydroxide) followed by the two-electron oxidation of the catechol to the ortho-quinone, which is first step in melanin biosynthesis. A unique feature of this enzyme is that the oxygen intermediate is stable in the absence of substrate, allowing it to be studied directly.

Extradiol dioxygenase. Extradiol dioxygenases are a large class of bacterial enzymes that function in the catabolism of aromatic carbon sources. They use a single Fe(II) ligated by two His, one Glu, and two waters in a square pyramidal geometry to cleave catechols between the 2- and 3- positions to yield 2-hydroxy-6-oxohexa-2,4-dienoic acids with the incorporation of both atoms from O₂. They are part of an immense, highly diverse family of non-heme iron oxygenases that are found everywhere from bacteria to humans. An unusual feature of extradiol dioxygenase is that is uses a single Fe(II) and O₂ to cleave an aromatic ring via a putative superoxide-Fe(II)-substrate radical.

Recent Publications

Lin IJ, Gebel EB, Machonkin TE, Westler WM, Markley JL (2005) Changes in hydrogen-bond strengths explain reduction potentials in 10 rubredoxin variants. Proc Natl Acad Sci U S A, 102:14581-6

Machonkin TE, Westler WM, Markley JL (2005) Paramagnetic NMR spectroscopy and density functional calculations in the analysis of the geometric and electronic structures of iron-sulfur proteins. Inorg Chem, 44:779-97

Knauf PA, Law FY, Leung TW, Atherton SJ (2004) Relocation of the disulfonic stilbene sites of AE1 (band 3) on the basis of fluorescence energy transfer measurements. Biochemistry, 43:11917-31

Machonkin TE, Westler WM, Markley JL (2004) Strategy for the study of paramagnetic proteins with slow electronic relaxation rates by nmr spectroscopy: application to oxidized human [2Fe-2S] ferredoxin. J Am Chem Soc, 126:5413-26

Lin IJ, Gebel EB, Machonkin TE, Westler WM, Markley JL (2003) Correlation between hydrogen bond lengths and reduction potentials in Clostridium pasteurianum rubredoxin. J Am Chem Soc, 125:1464-5

Machonkin TE, Westler WM, Markley JL (2002) (13)C[(13)C] 2D NMR: a novel strategy for the study of paramagnetic proteins with slow electronic relaxation rates. J Am Chem Soc, 124:3204-5

Machonkin TE, Markley JL (2002) Electron-Nuclear Interactions. in Encyclopedia of Nuclear Magnetic Resonance, Volume 9, edited by D.M. Grant and R.K. Harris, Wiley, 384

Machonkin TE, Quintanar L, Palmer AE, Hassett R, Severance S, Kosman DJ, Solomon EI (2001) Spectroscopy and reactivity of the type 1 copper site in Fet3p from Saccharomyces cerevisiae: correlation of structure with reactivity in the multicopper oxidases. J Am Chem Soc, 123:5507-17

Machonkin TE, Solomon EI (2000) The Thermodynamics, Kinetics, and Molecular Mechanism of Intramolecular Electron Transfer in Human Ceruloplasmin. J. Am. Chem. Soc., 122:12547

Machonkin TE, Musci G, Zhang HH, Bonaccorsi di Patti MC, Calabrese L, Hedman B, Hodgson KO, Solomon EI (1999) Investigation of the anomalous spectroscopic features of the copper sites in chicken ceruloplasmin: comparison to human ceruloplasmin. Biochemistry, 38:11093-102

Machonkin TE, Zhang HH, Hedman B, Hodgson KO, Solomon EI (1998) Spectroscopic and magnetic studies of human ceruloplasmin: identification of a redox-inactive reduced Type 1 copper site. Biochemistry, 37:9570-8

Graduate students in my laboratory work toward a Ph.D. degree in the following program[s]:

Ph.D. in Biophysics

Ph.D. candidates in my laboratory may also be affiliated with these programs:

Contact Information

E-Mail: Tim_Machonkin@urmc.rochester.edu

Timothy E. Machonkin
Department of Biochemistry and Biophysics
University of Rochester School of Medicine and Dentistry
601 Elmwood Ave, Box 712
Rochester, New York 14642

Office: Medical Center 3-7527
Telephone: (585) 275-0399; Fax: (585) 275-6007