Combined microspectrophotometric and crystallographic examination of chemically-reduced and X-ray radiation-reduced forms of cytochrome ba3 oxidase from Thermus thermophilus

摘要

Three paths are described to obtain crystals of reduced (II-E4Q/I-K258R) cytochrome ba3, and the structures of these are reported at ∼2.8 to 3.0 Å resolution. Microspectrophotometry of single crystals of Thermus ba3 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, U.S.A) being nearly complete in one minute. The previously reported structures of ba3 (PDB codes 1EHK and 1XME), having a crystallographically detectable water between the CuB and Fea3 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 CuB - Fea3 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 5-coordinate, low-spin, ferrous heme-a3 that is trapped in the frozen crystals.