Reaction mechanisms of non-heme diiron hydroxylases characterized in whole cells

Erin Bertrand, Ryo Sakai, Elena Rozhkova-Novosad, Luke Moe, Brian G. Fox, John T. Groves, Rachel N. Austin

Research output: Contribution to journalArticlepeer-review

34 Scopus citations


Whole cells expressing the non-heme diiron hydroxylases AlkB and toluene 4-monooxygenase (T4MO) were used to probe enzyme reaction mechanisms. AlkB catalyzes the hydroxylation of the radical clock substrates bicyclo[4.1.0] heptane (norcarane), spirooctane and 1,1-diethylcyclopropane, and does not catalyze the hydroxylation of the radical clocks 1,1-dimethylcyclopropane or 1,1,2,2-tetramethylcyclopropane. The hydroxylation of norcarane yields a distribution of products consistent with an "oxygen-rebound" mechanism for the enzyme in both the wild type Pseudomonas putida GPo1 and AlkB from P. putida GPo1 expressed in Escherichia coli. Evidence for the presence of a substrate-based radical during the reaction mechanism is clear. With norcarane, the lifetime of that radical varies with experimental conditions. Experiments with higher substrate concentrations yield a shorter radical lifetime (≈ns), while experiments with lower substrate concentrations yield a longer radical lifetime (≈19 ns). Consistent results were obtained using either wild type or AlkB-equipped host organisms using either "resting cell" or "growing cell" approaches. T4MO expressed in E. coli also catalyzes the hydroxylation of norcarane with a radical lifetime of ≈0.07 ns. No radical lifetime dependence on substrate concentration was seen. Results from experiments with diethylcyclopropane, spirooctane, dimethylcyclopropane, and diethylcyclopropane are consistent with a restricted active site for AlkB.

Original languageEnglish
Pages (from-to)1998-2006
Number of pages9
JournalJournal of Inorganic Biochemistry
Issue number10
StatePublished - Oct 2005

Bibliographical note

Funding Information:
We thank George Sisson, Charlotte Lehmann and Aubrey van Kirk for performing some of the experiments used in this analysis. We thank NSF CHE-0221978 (RNA, JTG) through the Environmental Molecular Sciences Institute CEBIC at Princeton University, NSF CHE-0116233 (RNA), the Camille and Henry Dreyfus Foundation (RNA), NIH GM072506 (RNA), NSF CHE-0316301 (JTG) and Bates College through a grant from the Hughes Foundation for support of this work. We thank Professor Gerben Zylstra and Dr. Hung-Kuang Chang for the generous gift of the E. coli TOP10-(pGJZ1371) clone.


  • AlkB
  • Alkane monooxygenase
  • Non-heme diiron hydroxylase
  • Radical clock substrates
  • T4MO

ASJC Scopus subject areas

  • Biochemistry
  • Inorganic Chemistry


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