Combined microspectrophotometric and crystallographic examination of chemically reduced and X-ray radiation-reduced forms of cytochrome ba 3 Oxidase from Thermus thermophilus: Structure of the reduced form of the enzyme

Bin Liu, Ying Chen, Tzanko Doukov, S. Michael Soltis, C. David Stout, James A. Fee

Research output: Contribution to journalArticlepeer-review

38 Scopus citations

Abstract

Three paths for obtaining crystals of reduced (II-E4Q/I-K258R) cytochrome ba 3 are described, and the structures of these are reported at ∼2.8-3.0 Å resolution. Microspectrophotometry of single crystals of Thermus ba 3 oxidase at 100 K was used to show that crystals of the oxidized enzyme are reduced in an intense X-ray (beam line 7-1 at the Stanford Synchrotron Radiation Laboratory), being nearly complete in 1 min. The previously reported structures of ba 3 (Protein Data Bank entries and), having a crystallographically detectable water between the Cu B and Fe a3 metals of the dinuclear center, actually represent the X-ray radiation-reduced enzyme. Dithionite-reduced crystals or crystals formed from dithionite-reduced enzyme revealed the absence of the above-mentioned water and an increase in the Cu B-Fe a3 distance of ∼0.3 A. The new structures are discussed in terms of enzyme function. An unexpected optical absorption envelope at ∼590 nm is also reported. This spectral feature is tentatively thought to arise from a five-coordinate, low-spin, ferrous heme a 3 that is trapped in the frozen crystals.

Original languageEnglish (US)
Pages (from-to)820-826
Number of pages7
JournalBiochemistry
Volume48
Issue number5
DOIs
StatePublished - Feb 10 2009
Externally publishedYes

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