Combined Microspectrophotometric and Crystallographic Examination of Chemically Reduced and X-ray Radiation-Reduced Forms of Cytochrome ba3 Oxidase from Thermus thermophilus: Structure of the Reduced Form of the Enzyme

摘要

Three paths for obtaining crystals of reduced (II-E4Q/I-K258R) cytochrome ba3 arendescribed, and the structures of these are reported at ∼2.8-3.0 Å resolution. Microspectrophotometrynof single crystals of Thermus ba3 oxidase at 100 K was used to show that crystals of the oxidizednenzyme are reduced in an intense X-ray (beam line 7-1 at the Stanford Synchrotron RadiationnLaboratory), being nearly complete in 1 min. The previously reported structures of ba3 (Protein DatanBank entriesnandn), having a crystallographically detectable water between the CuB and Fea3 metals of the dinuclearncenter, actually represent the X-ray radiation-reduced enzyme. Dithionite-reduced crystals or crystalsnformed from dithionite-reduced enzyme revealed the absence of the above-mentioned water and annincrease in the CuB-Fea3 distance of ∼0.3 A. The new structures are discussed in terms of enzymenfunction. An unexpected optical absorption envelope at ∼590 nm is also reported. This spectral featurenis tentatively thought to arise from a five-coordinate, low-spin, ferrous heme a3 that is trapped in thenfrozen crystals.