title: Photolysis of Hi-CO Nitrogenase - Observation of a Plethora of Distinct CO Species using Infrared Spectroscopy.
abstract: Fourier transform infrared spectroscopy (FT-IR) was used to study the photochemistry of CO-inhibitedAzotobacter vinelandiinitrogenase using visible light at cryogenic temperatures. The FT-IR difference spectrum of photolyzed hi-CO at 4 K comprises negative bands at 1973 cm-1and 1679 cm-1together with positive bands at 1711 cm-1, 2135 and 2123 cm-1. The negative bands are assigned to a hi-CO state that comprises 2 metal-bound CO ligands, one terminally bound, and one bridged and/or protonated species. The positive band at 1711 cm-1is assigned to a lo-CO product with a single bridged and/or protonated metal-CO group. We term these species 'Hi-1' and 'Lo-1' respectively. The high-energy bands are assigned to a liberated CO trapped in the protein pocket. Warming results in CO recombination, and the temperature dependence of the recombination rate yields an activation energy of 4 kJ mol-1. Two α-H195 variant enzymes yielded additional signals. Asparagine substitution, α-H195N, gives a spectrum containing 2 negative 'Hi-2' bands at 1936 and 1858 cm-1with a positive 'Lo-2' band at 1780 cm-1, while glutamine substitution, α-H195Q, produces a complex spectrum that includes a third CO species, with negative 'Hi-3' bands at 1938 and 1911 cm-1and a positive feature 'Lo-3' band at 1921 cm-1. These species can be assigned to a combination of terminal, bridged, and possibly protonated CO groups bound to the FeMo-cofactor active site. The proposed structures are discussed in terms of both CO inhibition and the mechanism nitrogenase catalysis. Given the intractability of observing nitrogenase intermediates by crystallographic methods, IR-monitored photolysis appears to be a promising and information-rich probe of nitrogenase structure and chemistry.
authors.affiliation: Department of Applied Science, University of California, One Shields Avenue, Davis, CA 95616 USA.
authors.forename: Lifen
authors.initials: L
authors.lastname: Yan
authors.affiliation: Department of Biochemistry, Virginia Polytechnic Institute and State University, 123 Engel Hall, Blacksburg, VA 24061, USA.
authors.forename: Christie H
authors.initials: CH
authors.lastname: Dapper
authors.affiliation: Department of Applied Science, University of California, One Shields Avenue, Davis, CA 95616 USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, MS 6R2100, Berkeley, CA 94720, USA.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: George
authors.affiliation: Department of Applied Science, University of California, One Shields Avenue, Davis, CA 95616 USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, MS 6R2100, Berkeley, CA 94720, USA.
authors.forename: Hongxin
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Applied Science, University of California, One Shields Avenue, Davis, CA 95616 USA.
authors.forename: Devrani
authors.initials: D
authors.lastname: Mitra
authors.affiliation: Department of Applied Science, University of California, One Shields Avenue, Davis, CA 95616 USA.
authors.forename: Weibing
authors.initials: W
authors.lastname: Dong
authors.affiliation: Department of Biochemistry, Virginia Polytechnic Institute and State University, 123 Engel Hall, Blacksburg, VA 24061, USA.
authors.forename: William E
authors.initials: WE
authors.lastname: Newton
authors.affiliation: Department of Applied Science, University of California, One Shields Avenue, Davis, CA 95616 USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, MS 6R2100, Berkeley, CA 94720, USA.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Cramer
keywords: Azotobacter vinelandii; CO; FT-IR; nitrogenase; normal mode; photolysis