title: A role for catalase-peroxidase large loop 2 revealed by deletion mutagenesis: control of active site water and ferric enzyme reactivity.
abstract: Catalase-peroxidases (KatGs), the only catalase-active members of their superfamily, all possess a 35-residue interhelical loop called large loop 2 (LL2). It is essential for catalase activity, but little is known about its contribution to KatG function. LL2 shows weak sequence conservation; however, its length is nearly identical across KatGs, and its apex invariably makes contact with the KatG-unique C-terminal domain. We used site-directed and deletion mutagenesis to interrogate the role of LL2 and its interaction with the C-terminal domain in KatG structure and catalysis. Single and double substitutions of the LL2 apex had little impact on the active site heme [by magnetic circular dichroism or electron paramagnetic resonance (EPR)] and activity (catalase or peroxidase). Conversely, deletion of a single amino acid from the LL2 apex reduced catalase activity by 80%. Deletion of two or more apex amino acids or all of LL2 diminished catalase activity by 300-fold. Peroxide-dependent but not electron donor-dependent kcat/KM values for deletion variant peroxidase activity were reduced 20-200-fold, and kon for cyanide binding diminished by 3 orders of magnitude. EPR spectra for deletion variants were all consistent with an increase in the level of pentacoordinate high-spin heme at the expense of hexacoordinate high-spin states. Together, these data suggest a shift in the distribution of active site waters, altering the reactivity of the ferric state, toward, among other things, compound I formation. These results identify the importance of LL2 length conservation for maintaining an intersubunit interaction that is essential for an active site water distribution that facilitates KatG catalytic activity.
authors: Department of Chemistry and Biochemistry, Auburn University , Auburn, Alabama 36849-5312, United States.
authors: Shalley N
authors: SN
authors: Kudalkar
authors: Olive J
authors: OJ
authors: Njuma
authors: Yongjiang
authors: Y
authors: Li
authors: Michelle
authors: M
authors: Muldowney
authors: N Rene
authors: NR
authors: Fuanta
authors: Douglas C
authors: DC
authors: Goodwin
mesh_terms: D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D006418:Heme; D010544:Peroxidases; C118175:catalase-peroxidase, Haloarcula; C081610:catalase-peroxidase, bacteria