B
iphas
ic gem
inate reb
ind
ing of CO to myoglob
in upon flash photolys
is has been assoc
iated tol
igand d
istr
ibut
ion
in hydrophob
ic cav
it
ies, structurally detected by t
ime-resolved crystallography,
xenonoccupancy, and molecular s
imulat
ions. We show that the t
ime course of CO reb
ind
ing to human hemoglob
inalso exh
ib
its a b
iphas
ic gem
inate reb
ind
ing when the prote
in
is entrapped
in wet nanoporous s
il
ica gel. As
imple branched k
inet
ic
scheme,
involv
ing the bound state A, the pr
imary dock
ing s
ite C, and a secondaryb
ind
ing s
ite B was used to calculate the m
icroscop
ic rates and the t
ime-dependent populat
ion of the
intermed
iate spec
ies. The act
ivat
ion enthalp
ies of the assoc
iated trans
it
ions were determ
ined
in the absenceand presence of 80% glycerol. Potent
ial hydrophob
ic dock
ing cav
it
ies w
ith
in the
images/g
ifchars/alpha.g
if" BORDER=0> and
images/g
ifchars/beta2.g
if" BORDER=0 ALIGN="m
iddle"> cha
ins ofhemoglob
in were
ident
if
ied by computat
ional model
ing us
ing
xenon as a probe. A hydrophob
ic pocket onthe d
istal s
ide of the heme, correspond
ing to X
e4 in Mb, and a nearby s
ite that does not have acorrespondence
in Mb were detected. Ne
ither potent
ial
xenon s
ites on the prox
imal s
ide nor a m
igrat
ionchannel from the d
istal to prox
imal s
ite was located. The small enthalp
ic barr
iers between states B and Care
in very good agreement w
ith the locat
ion of the
xenon s
ites on the d
istal s
ide. Furthermore, theconnect
ion between the two
xenon s
ites
is relat
ively open, expla
in
ing why the decreased mob
il
ity of theprote
in w
ith v
iscos
ity only sl
ightly perturbs the energet
ics of l
igand m
igrat
ion between the two s
ites.