title: Single-crystal resonance Raman spectroscopy of site-directed mutants of cytochrome c peroxidase.
abstract: Resonance Raman spectra are reported for single crystals of cytochrome c peroxidase (CCP) mutants, taken by using a microscope equipped with a variable-temperature stage. The spectra are similar to those observed for the mutant proteins in solution, but there are detectable differences having to do with the coordination and spin state of the heme. The Asn-235 mutant contains a mixture of six-coordinate high- and low-spin states with a detectably higher fraction of the former than in solution. Upon cooling even to 223 K, the heme is converted mostly to the low-spin form. The Phe-191 mutant likewise shows a high/low-spin six-coordinate mixture, together with a preponderant population of five-coordinate heme. Upon cooling, the high-spin six-coordinate population converts immediately to the low-spin form, while the five-coordinate population does so more slowly. This behavior is intermediate between that of native CCP and the Asn-235 mutant, consistent with an ancillary role for the normal Trp-191-Asp-235 H-bond in the proximal anchoring of the heme Fe. The Phe-51 mutant shows a dominant high-spin five-coordinate heme population in the single crystal, whereas in solution the six-coordinate form is dominant. This difference is mimicked by adding 2-methyl-2,4-pentanediol (MPD) to the solution and is attributed to the dehydrating effect of MPD, which is present during crystallization. Upon lowering the temperature, the five-coordinate heme converts partially to a six-coordinate high-spin form.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Dipartimento di Chimica, Università di Firenze, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Smulevich
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: J M
authors.initials: JM
authors.lastname: Mauro
authors.forename: J M
authors.initials: JM
authors.lastname: Wang
authors.forename: L A
authors.initials: LA
authors.lastname: Fishel
authors.forename: J
authors.initials: J
authors.lastname: Kraut
authors.forename: T G
authors.initials: TG
authors.lastname: Spiro
mesh_terms: D003578:Cytochrome-c Peroxidase / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman; D013696:Temperature
chemical_list: D003578:Cytochrome-c Peroxidase