title: Genetic analysis of the Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803 reveals subunit roles in association, assembly, maturation, and function.
abstract: Hydrogenases are metalloenzymes that catalyze 2H(+) + 2e(-) ↔ H(2). A multisubunit, bidirectional [NiFe]-hydrogenase has been identified and characterized in a number of bacteria, including cyanobacteria, where it is hypothesized to function as an electron valve, balancing reductant in the cell. In cyanobacteria, this Hox hydrogenase consists of five proteins in two functional moieties: a hydrogenase moiety (HoxYH) with homology to heterodimeric [NiFe]-hydrogenases and a diaphorase moiety (HoxEFU) with homology to NuoEFG of respiratory Complex I, linking NAD(P)H ↔ NAD(P)(+) as a source/sink for electrons. Here, we present an extensive study of Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803. We identify the presence of HoxEFUYH, HoxFUYH, HoxEFU, HoxFU, and HoxYH subcomplexes as well as association of the immature, unprocessed large subunit (HoxH) with other Hox subunits and unidentified factors, providing a basis for understanding Hox maturation and assembly. The analysis of mutants containing individual and combined hox gene deletions in a common parental strain reveals apparent alterations in subunit abundance and highlights an essential role for HoxF and HoxU in complex/subcomplex association. In addition, analysis of individual and combined hox mutant phenotypes in a single strain background provides a clear view of the function of each subunit in hydrogenase activity and presents evidence that its physiological function is more complicated than previously reported, with no outward defects apparent in growth or photosynthesis under various growth conditions.
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401, USA. carrie.eckert@nrel.gov
authors.forename: Carrie
authors.initials: C
authors.lastname: Eckert
authors.forename: Marko
authors.initials: M
authors.lastname: Boehm
authors.forename: Damian
authors.initials: D
authors.lastname: Carrieri
authors.forename: Jianping
authors.initials: J
authors.lastname: Yu
authors.forename: Alexandra
authors.initials: A
authors.lastname: Dubini
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Nixon
authors.forename: Pin-Ching
authors.initials: PC
authors.lastname: Maness
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D006864:Hydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046939:Synechocystis / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D006864:Hydrogenase