The present work has several implications for the chemistry of heme
proteins. (1) The existence of the PTM in GlbN [40] and its engineered
presence in GlbN [22] and CtrHb cautions that the PTM may occur in
additional heme proteins, not only wild-type but also unwittingly in
His-tagged versions, (histidine) variants, or designed proteins. (2) In preparing
the histidine Nε2–heme vinyl Cα crosslink, caremustbe exercised
in the choice of a reducing agent to avoid heme and protein damage, as
occasionally caused by the oxidative by-products of aerobic DT reduction.
AnaerobicDT treatment or reductionwith alternative agents such as a ferredoxin
system [56] if sufficiently powerful may be preferable. (3) It is
possible to engineer the crosslink in a b heme protein using relatively
mild reaction conditions. Although detailed kinetic analysis was not performed
on CtrHb variants, the products are consistent with the mechanism
described previously [21], in which case the pKa of the reactive
histidine, in addition to its ability to adopt the geometry of the product,
is an important factor. (4) The necessity of using the cyanomet adduct
as the species to be reduced rather than the ligand free protein (as in
GlbNs) is likely related to the conformation of the starting material. In
view of the CtrHb results, we expect that successful application to other
proteins will depend principally on the position of the engineered
histidine, the local flexibility of the supporting structure, the absence of
inhibiting distal ligands, and possibly the orientation of the heme
vinyl group.
The ability to prepare non-natively crosslinked versions of heme proteins
extends the range of heme chemistry questions that can be
addressed by biophysical methods. For example, towhat extent does protonation
of the alkylated histidine influence the heme reduction
potential? How do the histidine-heme linkages differ from the cysteine–
hemethioether linkages of c-type cytochromes?What is the electronic influence
of the distal ligand? Practical usage of the crosslinking reaction includes
the formulation of artificial enzymes or oxygen transporters with
non-dissociable heme; such systems are expected to have an elongated
lifetime, have increased resistance to proteolytic cleavage, andmay be capable
of functioning over a wide range of conditions (e.g. high temperature,
low pH) for industrial or biomedical applications.
The present work has several implications for the chemistry of hemeproteins. (1) The existence of the PTM in GlbN [40] and its engineeredpresence in GlbN [22] and CtrHb cautions that the PTM may occur inadditional heme proteins, not only wild-type but also unwittingly inHis-tagged versions, (histidine) variants, or designed proteins. (2) In preparingthe histidine Nε2–heme vinyl Cα crosslink, caremustbe exercisedin the choice of a reducing agent to avoid heme and protein damage, asoccasionally caused by the oxidative by-products of aerobic DT reduction.AnaerobicDT treatment or reductionwith alternative agents such as a ferredoxinsystem [56] if sufficiently powerful may be preferable. (3) It ispossible to engineer the crosslink in a b heme protein using relativelymild reaction conditions. Although detailed kinetic analysis was not performedon CtrHb variants, the products are consistent with the mechanismdescribed previously [21], in which case the pKa of the reactivehistidine, in addition to its ability to adopt the geometry of the product,is an important factor. (4) The necessity of using the cyanomet adductas the species to be reduced rather than the ligand free protein (as inGlbNs) is likely related to the conformation of the starting material. Inview of the CtrHb results, we expect that successful application to otherproteins will depend principally on the position of the engineeredhistidine, the local flexibility of the supporting structure, the absence ofinhibiting distal ligands, and possibly the orientation of the hemevinyl group.The ability to prepare non-natively crosslinked versions of heme proteinsextends the range of heme chemistry questions that can beaddressed by biophysical methods. For example, towhat extent does protonationof the alkylated histidine influence the heme reductionpotential? How do the histidine-heme linkages differ from the cysteine–hemethioether linkages of c-type cytochromes?What is the electronic influenceof the distal ligand? Practical usage of the crosslinking reaction includesthe formulation of artificial enzymes or oxygen transporters withnon-dissociable heme; such systems are expected to have an elongatedlifetime, have increased resistance to proteolytic cleavage, andmay be capableof functioning over a wide range of conditions (e.g. high temperature,low pH) for industrial or biomedical applications.
การแปล กรุณารอสักครู่..