molecular hydrogen ( h2 ) is a zero - emission energy carrier and is one of the best alternative fuels for the future , especially in urban areas with stricter environmental requirements .
hydrogenase ( h2ase ) enzymes catalyze the bidirectional reaction of 2h + 2e  h2 ( jugder et al . , 2013  ; kim & cha , 2013  ) and
thus have attracted interest for potential applications in h2 production in addition to their importance in biological sciences ( mertens & liese , 2004  ; cammack et al . , 2001  ; heinekey , 2009  ; tard & pickett , 2009  ; yang et al .
, 2011  ; fritsch et al . , 2013  ; simmons & artero , 2013  ; matsumoto et al . , 2013  ) .
while both diiron ( [ fefe ] ) and nickel  iron ( [ nife ] ) h2ases possess high catalytic activity ( evans et al .
, 2013  ; armstrong , 2009  ; shafaat et al . , 2013  ; lubitz et al . ,
2014  ) , the latter are attractive practically since they exhibit greater o2-tolerance ( guiral et al . , 2006  ; evans et al . , 2013  ; lauterbach et al . , 2015 
[ nife ] h2ase contains several fe  s clusters and one nife active site , in which fe is coordinated by one co and two cn ligands ( kamali et al .
the ni is coordinated to the protein matrix by four cysteinyl thiolates ( s ) , two of which serve as bridging ligands to fe .
in addition to these two s ligands , there could be a third ligand to bridge the two metal atoms in several enzymatic states ; for example , a hydroxide ligand in the oxidized inactive forms ( ni  a and ni  b ) ( dole et al . , 1997  ; gu et al . , 2003  ; gastel et al .
, 2015  ) and a possible hydride ( ni  h  fe ) in the active forms ni  c and ni 
1(b 
 1)1(b 
 6 )  ] , and the possible mechanism for h2 binding and cleavage at the h2ases active site .
for example , via a density function theory ( dft ) calculation , the structures in figs .
1(b 
 5 ) and 1(b 
 6 )  are believed to be the most likely candidates in the real ni 
information about the mechanism in turn could be useful for better producing h2 in the future . despite progress in characterization of h2ases by crystallography , infrared ( ir ) spectroscopy ( lubitz et al .
, 2014  ; fontecilla - camps et al . , 2007  ; de lacey et al . , 2007  ) and other x - ray spectroscopies ( wang et al .
, 2000  ; wang , patil , gu et al . , 2001  ; wang , patil , ralston et al . , 2001  )
, questions remain about the detailed molecular and electronic structure of various intermediates and inhibited species .
these highly debated questions include the nature or even the existence of a hydride bridge ( ni  h  fe ) in ni 
recent high - resolution crystallography demonstrated that the electron density as a hydride was detected at the bridging position between ni and fe ( ogata , nishikawa & lubitz , 2015  ) .
r represents a special challenge for spectroscopic studies for a number of reasons : ( i ) it is epr ( electron paramagnetic resonance ) silent ; ( ii ) the raman spectroscopy presents a problem due to its photoreactivity ; and ( iii ) ir spectroscopy for hydride bands is extremely difficult to observe ( jayapal et al .
thus there has been no progress in traditional vibrational spectroscopy so far and the existence of ni  h  fe in ni 
nuclear resonance vibrational spectroscopy ( nrvs ) scans an extremely monochromatic ( 1  mev ) x - ray beam through the nuclear resonance ( at 14.4  kev for fe ) , and measures the corresponding creation or annihilation of phonons ( seto et al . , 1995  ; sturhahn et al .
it is a relatively new x - ray spectroscopy that became available due to the development of third - generation synchrotron sources and advanced x - ray optics .
it has several distinguished advantages in comparison with traditional vibrational spectroscopic techniques such as ir and raman spectroscopies ( smith et al .
, 2005  ; kamali et al . , 2013  ; pelmenschikov et al . , 2011  ) . in the past ten years
, this technique has revealed / resolved fe  s / p / cl and fe  co / cn / no vibrational modes inside various inorganic complexes and iron enzymes and has become an excellent pin - point tool in recent years to study iron - specific inorganic and bioinorganic systems ( smith et al .
, 2005  ; cramer et al . , 2007  ; tinberg et al . , 2010 
in addition , fe  h - related nrvs features have also been resolved in several inorganic complexes ( bergmann et al .
via a series of careful and strenuous measurements , we have revealed the first direct spectroscopic evidence for ni 
r form of desulfovibrio vulgaris miyazaki f ( or dvmf ) [ nife ] h2ase using fe - specific nrvs . while its biochemical science and theoretical simulations have been presented and discussed recently ( ogata , kramer et al .
, 2015  ) , the measurement details , especially the strenuous experimental journey leading to the successful observation of the weak ni 
fe in a [ nife ] h2ase sample , and the task will face great difficulties due to the following two aspects : ( i ) a fe  h - related bending or wagging mode has a much weaker nrvs signal than fe  co ( pelmenschikov et al .
, 2011  ) while fe  co in nife h2ase is already rather weak ( kamali et al . , 2013  ; ogata , kramer et al . , 2015
( ii ) as will be discussed in detail later , its real energy position is far from either model complexes or theoretical predictions and was virtually unknown before it was observed experimentally .
this article evaluates all the matters related to whether the feature is observable , where to search for a fe  h - related feature , whether the observation is reliable , and how the experiments identify the observed 675  cm peak as a fe  h - related feature .
r nrvs spectra were recorded at spring-8 beamlines bl09xu ( yoda et al . , 2001  ) and bl19lxu ( yabashi et al .
bl09xu is a dedicated nuclear scattering beamline , and its instrumentation and properties have been discussed in detail elsewhere ( yoda et al .
, it has a si(111 ) double - crystal high - heat - load monochromator ( hhlm ) producing 14.4  kev radiation with 1.0  ev resolution , followed by a high - energy - resolution monochromator ( hrm ) [ ge(422 )  2si(975 ) ] producing 14.4  kev radiation with 0.8  mev resolution .
the beam flux was 1.4  10  photons s at 0.8  mev energy resolution .
the beam size was about 0.6  mm ( height )  1  mm ( width ) .
bl09xu has a dedicated nrvs measurement system , including a 2    2 avalanche photodiode ( apd ) detector array , a set of data - acquisition electronics and a control computer with data - acquisition software .
it also has a dedicated liquid - helium ( lhe ) flow cryostat for measuring nrvs at a cryogenic temperature for air- or temperature - sensitive samples , such as h2ases .
however , due to the extremely short distance from the samples surface to the apd array , a cold - finger cryostat has to be used instead of a more efficient gas exchange cryostat ( dong et al .
the real sample temperatures were much higher than 69  k , e.g. 5070  k ( wang et al . , 2012  ) .
this standard nrvs setup has been used in many nrvs measurements in the past and their details are discussed elsewhere ( cramer et al .
, 2001  ) provides the fundamental beam in the 7.218  kev energy region , covering fe nuclear resonance at 14.4  kev . as shown in fig .
2(a )  , it has a 25  m - long undulator ( hara et al . , 2002  ) instead of the usual 5  m - long undulator at spring-8
. therefore its hhlm provides 2.5  10  photons s at 14.4  kev , i.e. about five times higher than the beam intensity at bl09xu ( 5  10  photons  s after hhlm ) .
this beamline has many advanced applications and publications over a broad area from time - resolved measurements to weak feature observations ( tanaka et al .
it is also a good choice for nrvs measurements on weak vibrational features , which requires high beam intensity .
however , bl19lxu is not a dedicated nuclear scattering beamline and it does not have a fixed hrm and nrvs measurement station .
2b 
   ) which consists of a hrm , a lhe cryostat , a 2    2 apd detector array , a rack of associated electronics , and a control computer with data - acquisition software .
while these items are duplicates of those used at bl09xu , they need to be integrated into bl19lxu s experimental hutch 1 within about 36  h ( including optimization time ) .
therefore , bl19lxu has similar hhlm and hrm as those at bl09xu , and provided 14.4  kev radiation with 0.8  mev resolution , which is suitable for nrvs measurement .
the beam size was also 0.6  mm ( height )  1  mm ( width ) , the same as that at bl09xu .
after the hrm , a maximum 4.2 higher beam flux was recorded in comparison with that for bl09xu . however , owing to various practical limitations as well as using a moved - in hrm and nrvs apparatus , the ratio in nrvs counts per second ( cts / s ) for the two beamlines is 2.53.4 instead , while for bl09xu the cts / s is stable .
for example , in comparison with the bl09xu standard , the bl19lxu beam time for measuring the two ni 
r in h2/h2o ( nir - h for short ) samples in this study has a bl19/bl09 cts / s ratio of 2.6 , while that for measuring the ni 
r in d2/d2o ( nir - d for short ) has a ratio of 3 .
, 2007  ; seto et al . , 1995  ; sturhahn et al . , 1995  ; yoda et al . , 2001  ) , with a step size of 0.28  mev at bl09xu and 0.27  mev at bl19lxu .
the difference is due to minor difference in energy scales for the different beamlines ; the raw step sizes before the energy calibrations were both 0.295  mev .
the 14.4  kev beam with an energy resolution of 0.8  mev was scanned through the defined energy region ; the delayed nuclear fluorescence and fe k 
  fluorescence from fe were detected with the 2    2 apd array , processed by the electronics , and stored / displayed via the control computer .
the scanning region covered from 30  mev ( 240  cm ) to 70125  mev ( 5601000  cm ) depending on the different samples used and the different search cases .
, 1995  ) , where the observed raw nrvs spectra ( cts ) were calibrated to the nuclear resonance peak position , normalized to i 
 0 , summed and converted to the single - phonon fe partial vibrational density of states ( pvdos for abbreviation ) .
the spectral conversion was optimized when the observed stokes / anti - stokes imbalance matched the imbalance calculated using the entered temperature as a parameter .
the real sample temperatures were thus obtainable with the measured nrvs s stokes / anti - stokes imbalance ( wang et al . , 2012  ) .
the energy position for each scan was calibrated by aligning the elastic peak to zero during the nrvs spectral analysis with phoenix .
the energy scales were usually calibrated with a standard sample of [ fecl4][net4 ] with a prominent peak at 380  cm .
alternatively , the energy scales can also be calibrated with fe metal powder at the quick - switch calibration stage at the back of the main measurement stage ( wang et al .
it has a clear peak at 287  cm at room temperature . the background noise level ( dark current cts / s )
was estimated prior to ( and sometimes during ) each beam time by tuning the hrm s energy position to about 100  mev or about 800  cm ( in reference to the resonance elastic energy ) , where no vibrational peak is present .
signal was accumulated for either 500  s in total or five consecutive 100  s to obtain an average cts / s . for almost all the beam times , the dark current was 0.03  cts / s for our nrvs measurement system at bl09xu and at bl19lxu . since the relative strength of nrvs transitions varies dramatically and since it is necessary to emphasize one region of interest ( e.g. for searching for ni 
fe ) , the scans were divided into segments with different data collection times ( seconds per point , or s / p ) . in general , 13 
s / p was used for the range from 240 to 400  cm ( covering the fe  s region ) , then 510  s / p for the fe 
cn and fe  co region from 400 to 620  cm . a longer scanning time ( 1030  s ) was used for the candidate ni  h  fe searching region ( e.g. at 620770  cm ) .
this practice is used for the nrvs measurements at both bl09xu and bl19lxu , but the exact timing for different beam times varied a little .
the scan details will be discussed again in the results and discussions section . to compare the nrvs data from bl09xu and bl19lxu
, we re - scale the bl19lxu counting time based on its maximum cts / s versus the standard maximum cts / s at bl09xu and create bl09xu equivalent seconds , e.g 10  s / p at bl19lxu corresponds to 26 or 30 equivalent s / p at bl09xu [ corresponding to a cts / s ratio of 2.6 for measuring nir - h and 3 for measuring nir - d ] .
nir - h was measured once at bl09xu and twice at bl19lxu with an equivalent bl09xu time of 10  680 = 680  s / p , 20  24  2.6 = 1248  s / p and 15  12  2.6 = 468  s / p , respectively , in total 2396  s / p ( with a percentile of 28% , 52% and 20% ) .
nir - d was measured three times at bl09xu and once at bl19lxu , equivalent to 3980 bl09xu s / p [ = ( 40  20 ) + ( 30  30 ) + ( 36  30 ) + 20  20  3 = 2780 + 400  3 = 3980 ] in the same region . more s / p was used for nir - d because more time should be used to conclude that there is no signal than to find a weak signal .
fermentation was carried out in a 10  l glass fermenter under anaerobic conditions , and the [ nife ] h2ase expressed was isolated and purified as described earlier ( ogata et al .
for preparing nir - h ( nir - d ) , the as - isolated [ nife ] h2ase was transferred from 25  mm tris - hcl ( ph = 7.4 ) buffer to 100  mm mes ( ph = 5.0 or pd = 5.0 ) and was then purged with 1.2  bar h2 ( or 1.3  bar d2 ) for more than 8  h. the solutions were then loaded into nrvs cells under anaerobic conditions .
fourier transform ir ( ftir ) spectra were recorded on a bruker ifs66v / s ftir spectrometer to ensure the samples were in their supposed states ( ni  r1 : one of the subforms of ni 
it was measured in transmission mode in a sealed ir cell at room temperature and with a 2  cm energy resolution .
nrvs or the fe  h / d - related bending positions for several fe 
, 2011  ; crossland et al . , 2009  ; schilter et al . , 2012
 ) : ( i ) [ feh(d)6][mgbr(thf)2]4 ( or fefeh6/d6 for short ) ; ( ii ) fe(h / d)(co)(dppe)2 , dppe = 1,2-bis(diphenylphosphino)ethane ( or h / dfeco ) ; ( iii ) trans-[fe(dmeoprpe)2(n2)h ] { dmeoprpe = 1,2-bis[bis(methoxypropyl)phosphino]ethane } ( or hfen2 ) ; ( iv ) [ ( ,k2-bdt - h)(-pph2)(-h)fe2(co)5][otf ] ( or fehfe ) ; ( v ) [ ( dppe)ni(-pdt)(-h)fe(co)2(ph3p ) ] ( * = nature abundant , dppe = 1,2-ph2pch2ch2pph2 , pdt = sch2ch2ch2s ) ( or nihfex ) ; ( vi ) [ ( dppe)ni(-pdt)(-h / d)fe(co)3 ] ( or nih / dfe ) .
in addition , [ fecl4][net4 ] and metal fe were used as calibration samples for energy scales ( smith et al . , 2005  ;
the overall nrvs spectra for dvmf nir - h ( blue ) and nir - d ( red ) are presented in fig .
the nrvs spectra include the very low energy intensities from the backbone motion , the 100400  cm peaks for fe  s cluster vibrations , the 420530  cm peaks for fe 
cn modes , the 530630  cm peaks for fe  co modes ( lauterbach et al . , 2015  ; kamali et al . ,
2013  ) and a weak but clear peak at 675  cm , which are assigned to the ni  h 
3  , the top bars indicate different vibrational regions while the bottom bars illustrate the total time ( s ) used to measure the nir - h ( blue text ) and nir - d ( red text ) in each region . the blue text in the middle shows the signal level ( cts / s ) for each of the various signature peaks . while these middle blue numbers illustrate the signal level ( cts / s ) for nir - h , both nir - h and nir - d have about the same signal level .
co / cn features have a much lower cts / s than the fe  s features while the ni  h  fe has an even lower cts / s in comparison with the weak fe 
this provides readers with a basic idea of how weak the ni  h  fe peak in ni 
1(b 
 1)1(b 
 6 )  ] ( ogata , kramer et al . ,
2015  ) , the existence of ni  h  fe at 675  cm for nir - h ( blue ) and its absence for nir - d ( red ) provided a critical observation reference for the dft calculations , not the other way around . without this successful observation ,
r is observable or not . as per the nrvs statistics at bl09xu between 2006 and 2010 , when the hrm s energy resolution is at 0.9  mev , 1  mm of fe sample produced a 30  cts / s signal in the elastic peak ( wang et al .
4  , using 30  cts / s per 1  mm 
 fe .
the first two columns describe vibrational feature names and their vibrational energy positions , and the third column presents the nrvs signals for a hypothetical 1  mm h2ase ( or 12  mm 
 fe ) sample , which is similar to the h2ase measured in the past ( kamali et al .
it has 360  cts / s in the resonance peak , 0.71.2  cts / s for the fe  s peaks ( at 150380  cm ) , and 0.06  cts / s signal for fe  co ( at 600  cm ) .
co features were successfully observed with a 1  mm h2ase sample in the past ( kamali et al .
fe was too weak to be observed under previous conditions ( 1  mm h2ase concentration ) , so we have to estimate its potential cts / s ( the numbers below the
 fe and fe  co in available complexes , e.g. 1/5 for nihfe ( fig .
5b ) . a value of 0.012  cts / s is estimated for ni  h
 fe in 1  mm nir - h at bl09xu ; it is under the dark cts / s level ( 0.03  cts / s ) and is not observable under the given circumstances . since the completion of the nrvs measurements for kamali et al.s publication ( kamali et al .
, 2013  ) , further progress has been made to increase the potential nrvs signal level ( cts / s ) .
the major advance is the samples concentration increasing from 1  mm to 4  mm , which puts the ni 
fe feature at a hypothetical level of 0.012  4 = 0.048  cts / s .
some other minor improvements also contribute to the increase of cts / s : ( i ) bl09xu s hrm advanced from 0.9  mev to 0.8  mev energy resolution with almost the same level of beam intensity , which led to an increase in peaks cts / s [ maximum = ( 0.9/0.8 ) = 112.5% ] ; ( ii ) due to better control of the sample temperature during the nrvs measurement ( wang et al .
, 2012  ) , we are able to move the sample even closer to the apd .
it is difficult to estimate the real movement of the sample position but we obtained about 10% signal level increase in cts / s due to sample position alternation while still maintaining the sample temperature below 70  k. these two points thus mean all the nrvs peaks have 1.2 height .
fe is then about 0.048  1.2 = 0.058  cts / s , which is comparable with the fe  co feature for 1  mm h2ase in the previous measurement ( kamali et al .
4  ( not counting the narrow columns showing the arrow symbols ) shows the estimated cts / s for a hypothetical 4  mm h2ase , which will be measured at the improved bl09xu ( 1.2 ) .
then , how much time is needed to reveal such a weak peak ( 0.058  cts / s ) with a reasonable signal - to - noise ratio ( s / n ) ? in a previous publication ( wang et al .
, 2014  ) we have concluded that a 10  mm 
 fe sample has 1  cts / s in the fe  s region and 100  s / p is thus enough for a s / n = 100 = 10 . using the same principle
fe can lead to 0.058  500 = 29 pure cts and s / n = 29 = 5.4 .
although dark - current cts / s limits the best possible s / n , as will be discussed in detail later , an as - high - as - possible statistical s / n is still required and thus 500  s / p is a kind of realistic minimum time required to achieve a reasonable s / n for a weak feature like ni  h  fe . as a reference , the very weak fe  co features for the oxidized and the reduced 1  mm h2ase were observed with 580 and 2180  s / p total scanning time at bl09xu in the previous publication ( kamali et al . , 2013  ) . the 500  s / p scanning time means , for example , 25  20  s / p , leading to about 24  h or more beam time for one measurement with one ni 
fe signal is as important as ( if not more important than ) knowing it is observable .
r , which requires long ( e.g. 24  h or more ) beam times for each search .
since no nrvs features for iron hydrides or iron deuterides in a real iron enzyme have been published before this work , we used nrvs for a series of iron hydride / deuteride complexes and their fe  h - related vibrational positions ( fig . 5  ) as a starting point to discuss the possible location of the ni 
the nrvs feh6 ( blue)/fed6 ( red ) spectra were among the first nrvs spectra obtained for chemical complexes ( bergmann et al .
their features include : the x  fe  h / x  fe  d bending modes at 790/572  cm and the fe 
later on , many other iron - hydride - containing complexes were published or evaluated , e.g. the ( h / d)feco model complexes have the x  fe  h / x  fe  d at 740/620  cm , respectively ( pelmenschikov et al . , 2011  ) .
we noticed that the x  fe  d bending peak for this complex is close to the fe 
the nihfe complex is used to model [ nife ] h2ases active site and it mimics the structure for the [ nife ] center with very minor structural differences ( barton et al . , 2009  ; shafaat et al .
its ni  h  fe wag mode is at 758  cm ( fig .
d stretching ( mixed with fe ) per dft calculation ( ogata , kramer et al .
it is not fe  d - related bending , which is mixed into the fe  co features .
the nrvs feature most relevant to ni  r s ni  h  fe is the x  fe  h or ni  h  fe feature in various complexes .
the energy positions of these fe  h - related bending modes for feh6 , hfeco , hfen2 , fehfe , nihfex and nihfe ( refer to  2 for the full names of these complexes ) are illustrated in fig .
5(b ) . the blue shaded area highlights the possible fe  h - related bending energies while the orange ( purple ) area indicates the fe 
d bending ( stretching ) mode regions . from these fe  h / d complexes
we have learned the following : ( i ) although fe  d - related bending has more intensity than fe  h - related bending , it may mix with fe  co and become unresolvable .
therefore , for fe  co - containing complexes , the fe  h - related bending mode has the highest observable nrvs peak among all of the fe  h / d - related stretching and bending modes .
( ii ) the fe  h - related bending mode positions span from 740 to 790  cm for the examined iron hydride complexes , including those whose spectra are not shown here .
dft was also used to make a prediction about the possible position of this critical vibrational peak .
however , all of the earlier dft calculations as well as normal mode analyses ( before the ni 
fe was finally observed experimentally ) pointed to a region with energies higher than 760  cm ; some of them suggested a region higher ( or much higher ) than 800  cm and we experimentally searched up to 125  mev ( or 1000  cm ) , but did not find anything .
fe regions was searched one to two times but led to no success at all .
although 620720  cm became the only region not searched with a great investment of beam time , the idea to search in this region was not at all popular at the time , because all the model complexes and theoretical calculations pointed to a much higher energy position .
r model complex nihfe has its ni  h  fe at 758  cm , 140  cm higher than its highest - energy fe  co peak at 617  cm , while ni 
then the question arose : should we perform a thorough search in this unlikely but unsearched region ?
fe at 675  cm . as searching in the 620720  cm region was still not a mainstream opinion at that time , we first performed a balance measurement between acquiring a perfect fe  co spectrum and searching for ni 
fe with a 4.5  mm nir - h sample at ( improved ) bl09xu .
therefore an even scanning time of 10  s / p was used between 400 and 740  cm instead of spending more time ( e.g. 2030  s ) in the ni 
( with a total scanning time of 680  s / p ) a weak but positive peak at 675  cm was observed for the first time as shown in fig .
6(a )  , with the raw nrvs spectrum at the top and the converted pvdos at the center ( 10 ) and the bottom ( 1 ) .
we noticed that the 675  cm energy position is much lower than any of the previous dft or normal mode analyses  predicted theoretical energy positions .
it is actually still at least 32  cm lower than the lowest current dft value , which is at 707  cm [ model vi in fig .
1(b 
 6 )  ] ( ogata , kramer et al . , 2015  ) .
the observed cts / s for various fe  s , fe  co and ni  h  fe in ni 
r are documented in fig . 4  [ column 6 ( not counting the columns showing 
the first argument was that the observed peak at 675  cm was still too weak .
the second argument attributed this peak to a fe  co - related feature , rather than to a fe  h - related peak .
however , there is no such fe  co sub - feature in the mimic complex nihfe ( ogata , kramer et al .
these nrvs measurements on nir - d took 106 scans in total (= 40 + 30 + 36 ) and 2780 total s / p [ = ( 40  20 ) + ( 30  30 ) + ( 36  30 ) ] , but concluded no peak in the same region .
this suggests that the peak at 675  cm is related to a fe  h vibrational mode . in order to resolve the first issue ( to repeat the observation and to improve the spectral s / n ) and to further clarify the second issue , two samples of nir - h and one sample of nir - d were re - measured at bl19lxu , which has a higher beam intensity and more nrvs cts / s than bl09xu .
a similar pair of nrvs and pvdos for nir - h ( blue , 24 scans , 480  s / p ) and for nir - d ( red , 20 scans , 400  s / p ) are compared in fig .
6(b )  , illustrating much clearer evidence for the existence and the fe  h - related nature for the 675  cm peak . from fig . 4  ( column 6 for one measurement at bl19 and column 7 for one measuremnt at bl19 , not counting the columns with the arrow symbols ) , the observed weak ni 
fe wagging mode is 2729 parts per million in intensity in comparison with the nuclear resonance peak , or 0.8% in comparison with the fe 
co ( s ) , this ratio is about 14.816.6% , or , equivalently , ni  h  fe : fe  co  1:61:7 .
for such a weak signal , the apd s dark - current noise ( instrument error ) defines the best possible s / n because the instrumental error bar can not be averaged lower with more signal statistics .
therefore a low ( 0.03  cts / s ) dark current is the most important foundation for a successful observation of weak ni 
fe for a hypothetical 4  mm nir - h but measured at bl19lxu , which has 2.6 times more cts / s , is listed in column 5 of fig .
the real signal level for a 4.1  mm nir - h at bl19 is listed in column 7 : ni  h  fe = 0.12  cts / s for measurement 1 [ and 0.13  cts / s for measurement 2 ( not listed ) ] . in comparison with the hypothetical case ( column 5 ) , the real measurement ( column 7 ) of ni  h 
fe has a slightly lower cts / s level although the corresponding fe  co has a higher cts / s level .
this is because ni  h  fe : fe  co is about 1:6 for nir - h ( fig . 
nir - h was measured once at bl09xu ( 680  s ) and twice at bl19lxu ( 480 + 180  s ) .
the signal level at bl09xu is estimated at ( 94  60)/680 = 0.05  cts / s , leading to a s / n = 0.05/0.03  1.7 and a not - so - clear nrvs ( fig .
6b 
   ) is ( 139  81)/480 = 0.12  cts / s , corresponding to a s / n = 0.12/0.03 = 4 , a much clearer observation for the ni 
the signal level for measurement 2 at bl19lxu is about 0.13 and s / n = 4.3 .
the total equivalent bl09xu s / p ( as defined in the experimental section ) for nir - h in the nifeh region is 2396  s and the averaged signal level is 0.1  cts / s . assuming as - high - as - possible statistics , this leads to s / n = 3.3 . as the real statistics are not infinite , the real s / n is slightly lower than 3.3 .
nir - d was measured three times at bl09xu and once at bl19lxu , equivalent to 3980 bl09xu s / p in the same region ( versus 2396  s for nir - h ) . as there is no peak
, there is no meaning to estimating the signal level or s / n for nir - d .
however , a more equivalent s / p was used for nir - d because more time should be used to conclude that there is no signal than to find a weak signal .
7  summarizes three pvdos for nir - h and four pvdos for nir - d in the region around 675  cm .
again , all nir - h pvdos show a clear peak around 675  cm while none of those for nir - d show a peak in the same region , providing solid evidence that the observed 675  cm peak is real and is related to fe 
there are several experimental conditions which make this work successful and a super - concentrated ( 4  mm ) and almost pure ni 
r ) is one of the most important conditions but is also difficult to realise .
we proceed with exchange ph of the buffer ( 7.4  5.0 ) and with a longer period ( more than 8  h ) of h2/d2 reduction . monitoring samples integrity can never be more important than under these circumstances .
the following three steps were used to monitor the samples integrity : ( i ) nir - h / d were first checked with ftir at the sample preparation laboratory before sending to spring-8 for nrvs measurements , as shown in fig .
these subforms are distinguishable by ftir and differ in their protonation states but they are all ni  r .
r1 ( co = 1946  cm ) , 16% nic ( at 1962  cm ) and a trace amount of ni  r2 ( co = 1933  cm ) .
( ii ) nir - h / d after nrvs measurements were also collected and checked with ftir and both ir spectra [ blue for nir - h and red for nir - d in fig .
8(b )  ] show little change in comparison with those measured before sending for nrvs experiments ( fig .
8a 
   ) , confirming the integrity of the samples for the whole nrvs measurement . (
iii ) immediately prior to a nrvs measurement , in situ nrvs measurement in a sample s fe 
s region was another way of carrying out a last - minute check , as shown in fig .
9 . for example , the light blue curve shows one example of many good ni 
7  ) , while the black curve shows one bad ( unexpectedly oxidized )  ni 
the green and red spectra are the established nrvs for the oxidized and reduced dvmf h2ase ( kamali et al . , 2013  ) .
r , the real control over the samples integrity is via the ftir measurements prior to and following the nrvs experiments , as shown in fig .
8 . this in situ examination was used only as a handy check that the sample was not oxidized ( e.g. fig .
9  , black curve ) before investing a long beam time on the sample .
there are a lot of features in the fe  s region which can be used to characterize the oxidation states of nife h2ase , including the peaks between 340 and 460  cm ( kamali et al .
. 4  , column 4 ) , a hypothetical 4  mm dvmf h2ase sample will have 36  cts / s in its fe  s region , and three regular 5  s / p scans will produce 4590 total cts or a statistical s / n of 6.79.5 .
as the dark current is much smaller in comparison with the fe  s signal cts / s , the statistical s / n is close to the real s / n .
then a s / n of 79 should be sufficient to resolve the samples oxidation states . at bl19lxu , in principle , one scan is enough , but we usually took two 3  s / p scans in order to check the repeatability .
we noticed that the low dark cts / s of the apd array ( i.e. 0.03  cts / s ) stays the same at bl19lxu , which has a higher beam intensity than bl09xu .
if this dark - current level increases at bl19lxu , the increased cts / s will become less meaningful .
we also noticed that the higher beam intensity at bl19lxu does not lead to higher sample temperatures ( wang et al .
this again illustrates the conclusion in our previous publication , which suggested that the hike in the samples temperatures in comparison with the sample base is mainly due to thermal radiation from the room - temperature window near the samples rather than from the x - ray irradiation ( wang et al .
, 2012  ) . as the hrm is inside the same hutch as the nrvs measurement chamber ,
entering the hutch can disturb the hutch temperature and the performance of the hrm . a sample change or other regular operation , which lasts 30  min , will lead to a system recovery time of about 2  h. in the case of an emergency entry for 1  min , the system can be recovered in about 2030  min . as mentioned earlier , the energy scale can be calibrated either normally or with a quick - switch calibration procedure ( wang et al . , 2013  ) .
for concentrated nir - h / d samples , the fe  s peak at 365  cm ( fig .
9  ) can also be used as a calibration reference because of its high signal level .
the fe  h or fe  d stretching modes will have no overlaps with any possible nrvs peaks in the same regions and will allow a straightforward experimental identification of the hydride .
however , in reference to the model complexes and dft calculations ( ogata , kramer et al .
, 2015  ) , fe  d has 1/5 of the intensity of ni  h  fe ( see fig .
5  ) while fe  h has 1/15 of that of ni  h  fe ( not shown in fig . 5  ) , presenting further challenges . under the current experimental conditions ,
all these peaks are still not observable unless significant improvements in beam intensity , sample concentration and/or measurement solid angles are made .
different improvements and tests are underway . from the nidfe complex , the 711  cm ni 
d stretching bending peak ( mixed with fe ) has about the same intensity as ni  h  fe .
however , it could be mixed with fe  co and is not observable in ni 
this publication has presented the strenuous experimental journey to discover the first spectroscopic evidence for the existence of a bridging hydride in a [ nife ] h2ase s ni 
fe wagging mode has 2729 parts per million in intensity in comparison with the nuclear resonance peak , or 0.8% in comparison with a fe  s feature .
improved sample concentration , increased photon flux and ultra - low noise level in the apd detectors all set the foundation for a possible nrvs measurement of the weak ni 
the decision and insistence on searching in the then unlikely energy region of 620720  cm was the key step towards successful observation of the ni 
the existence / non - existence of the 675  cm peak for nir - h / nir - d identified the peak as a fe  h - related feature experimentally .
fe feature at 675  cm provided a critical observed reference for dft calculations and thus served as a key in the dft determination of the ni 
it will be of interest to other scientists who use synchrotron radiation for measuring dilute samples and/or weak spectroscopic features in general .
 direct spectroscopic evidence for a hydride bridge in the ni  
 r form of [ nife ] hydrogenase has been obtained using iron - specific nuclear resonance vibrational spectroscopy . 
 this article focuses on the long and strenuous experimental journey to search for and identify this first spectroscopic evidence for a hydride in ni  
 r . 