Biogenesis of cytochromec oxidase (COX) is a highly complex process
involving 30 chaperones in eukaryotes; those required for the incorpora-tion of the copper and heme cofactors are also conserved in bacteria.
Surf1, associated with hemeainsertion and with Leigh syndrome if defec-tive in humans, is present as two homologs in the soil bacterium Paracoc-cus denitrificans, Surf1c and Surf1q.
The protonation state of residues around the Qo binding site of the cyto-chrome bc1 complex from Paracoccus denitrificansand their interaction
with bound quinone(s) was studied by a combined electrochemical and
FTIR difference spectroscopic approach. Site-directed mutations of two
groups of conserved residues were investigated: (a) acidic side chains
located close to the surface and thought to participate in a water chain
leading up to the heme bLedge, and (b) residues located in the vicinity of
this site. ...
The heterologous expression of tryptophan trytophylquinone (TTQ)-dependent aromatic amine dehydrogenase (AADH) has been achieved in
Paracoccus denitrificans. The aauBEDAgenes andorf-2from the aromatic
amine utilization (aau) gene cluster of Alcaligenes faecaliswere placed
under the regulatory control of themauFpromoter fromP. denitrificans
and introduced intoP. denitrificansusing a broad-host-range vector.
In recent studies on heme-copper oxidases a particular glutamate residue in
subunit II has been suggested to constitute the entry point of the so-called
K pathway. In contrast, mutations of this residue (E78
) in the Paracoccus
denitrificanscytochromecoxidase do not affect its catalytic activity at all
Q) or reduce it to about 50% (E78
A); in the latter case, the mutation
causes no drastic decrease in hemea3
reduction kinetics under anaerobic con-ditions, when compared to typical K pathway mutants....
Introducing site-directed mutations in surface-exposed residues of subunit II of the heme aa3 cytochrome c oxidase of Paracoccus denitriﬁcans, we analyze the kinetic parameters of electron transfer from reduced horse heart cytochrome c. Speciﬁcally we address the following issues: (a) which residues on oxidase contribute to the docking site for cytochrome c, (b) is an aromatic side chain required for electron entry from cytochrome c, and (c) what is the molecular basis for the previously observed biphasic reaction kinetics....
Tightly coupled inside-out vesicles were prepared from
Paracoccus denitrificanscells (SPP, sub-Paracoccusparticles)
and characterized kinetically. The rate of NADHoxidation,
catalysed by SPP, increases 6–8 times on addition of gram-icidin. The vesicles are capable of catalysing DlH
-dependent reverse electron transfer fromquinol toNAD
The kinetic parameters of the NADH-oxidase and the
reverse electron transfer carried out by membrane-bound
P. denitrificanscomplex I were estimated and compared
with those of the mitochondrial enzyme. ...
The Rieske [2Fe)2S] protein (ISP) is an essential subunit of cyto-chromebc1
complexes in mitochondrial and bacterial respiratory chains.
Based on the presence of two consecutive arginines, it was argued that the
ISP ofParacoccus denitrificans, a Gram-negative soil bacterium, is inserted
into the cytoplasmic membrane via the twin-arginine translocation (Tat)
Two soluble enzymes (FerA and FerB) catalyzing the
reduction of a number of iron(III) complexes by NADH,
were purified to near homogeneity from the aerobically
grown iron-limited culture ofParacoccus denitrificansusing
a combination of anion-exchange chromatography (Seph-arose Q), chromatofocusing (Mono P), and gel permeation
chromatography (Superose 12). FerA is a monomer with
a molecular mass of 19 kDa, whereas FerB exhibited a
molecular mass of about 55 kDa and consists of probably
two identical subunits. ...
The structure of cytochromec-550 from the nonphotosynthetic bacteria
Paraccocus versutushas been solved by X-ray crystallography to 1.90 A˚
resolution, and reveals a high structural homology to other bacterial cyto-chromesc2. The effect of replacing the axial heme-iron methionine ligand
with a lysine residue on protein structure and unfolding has been assessed
using the M100K variant.