Low frequency dynamics of the nitrogenase MoFe protein via femtosecond pump probe spectroscopy — Observation of a candidate promoting vibration

Abstract

We have used femtosecond pump-probe spectroscopy (FPPS) to study the FeMo-cofactor within the nitrogenase (N2ase) MoFe protein from Azotobacter vinelandii. A sub-20-fs visible laser pulse was used to pump the sample to an excited electronic state, and a second sub-10-fs pulse was used to probe changes in transmission as a function of probe wavelength and delay time. The excited protein relaxes to the ground state with a ~1.2 ps time constant. With the short laser pulse we coherently excited the vibrational modes associated with the FeMo-cofactor active site, which are then observed in the time domain. Superimposed on the relaxation dynamics, we distinguished a variety of oscillation frequencies with the strongest band peaks at ~84, 116, 189, and 226 cm−1. Comparison with data from nuclear resonance vibrational spectroscopy (NRVS) shows that the latter pair of signals comes predominantly from the FeMo-cofactor. The frequencies obtained from the FPPS experiment were interpreted with normal mode calculations using both an empirical force field (EFF) and density functional theory (DFT). The FPPS data were also compared with the first reported resonance Raman (RR) spectrum of the N2ase MoFe protein. This approach allows us to outline and assign vibrational modes having relevance to the catalytic activity of N2ase. In particular, the 226 cm−1 is assigned as a potential ‘promoting vibration in’ the H-atom transfer (or proton-coupled electron transfer) processes that are an essential feature of N2ase catalysis. The results demonstrate that high-quality room-temperature solution data can be obtained on the MoFe protein by the FPPS technique and that these data provide added insight to the motions and possible operation of this protein and its catalytic prosthetic group.

The Interstitial Carbon of the Nitrogenase FeMo Cofactor is Far Better Stabilized than Previously Assumed

“…With aq uantum chemical (BP86/def2-pvtz) averaged force constant of well above 1Ncm À1 (FeÀC inter : 1.31 Ncm À1 ,see Table 1), the carbon appears to be covalently bound in the resting state.E arly experimental nuclear resonance vibrational spectroscopy (NRVS) data from Xiao et al [18] evaluated the strength of the Fe À C inter interaction as being significantly below one Newton per centimeter (0.32 Ncm À1 ,T able 1), values that correspond rather to the binding strength in the high-spin state of the FeMo cofactor without any antiferromagnetic coupling (see the Supporting Information). [19] Thee lastic strength within the iron cluster itself is also,a ccording to our calculations,m uch higher than hitherto assumed:w ith an Fe À Fe force constant of 1.23 Ncm À1 (older, empirical NRVS force constant:0 .27 Ncm À1 ,s ee Table 1), these bonds are much stronger than previously thought. This signal might be useful as as upplement to the signature suggested by Pelmentschikov,C ramer, and coworkers of 226 cm À1 for further analysis of the prosthetic group of nitrogenase during catalysis.…”

Resonance Raman spectroscopy of Fe–S proteins and their redox properties

“…In fact the first RR spectrum of a nitrogenase from Azotobacter vinelandii that houses the ‘FeMo-cofactor’ in the active site (i.e., [Mo–7Fe–9S-Ci], where Ci is carbide), has been reported only recently, employing high laser power and exceptionally long accumulation times. The spectra obtained with 488 nm excitation reveal A 1 b mode at 338 cm −1 , together with two additional bands 356 and 382 cm −1 in nitrogenase [41]. …”

“…Then we will focus on the major contributions of RR spectroscopy in the studies of diverse Fe–S proteins, such as those that participate in ET [1, 13–21], DNA repair [11], biogenesis of Fe–S clusters [3, 22–30] and heme cofactors [31], substrate binding and activation, S-donation and catalysis [32–35], and regulation of gene expression [5, 8, 10, 36–39]. We will conclude with several examples of recent RR studies of enzymes carrying complex polychromophoric clusters [7, 40, 41]. …”

Der interstitiell gebundene Kohlenstoff der Nitrogenase ist deutlich stabiler als bisher angenommen

“…[18] Eine Analyse der Normalmoden und der Dipolmoment-Ableitungen lässt die Fe-C inter -Frequenz als Signal im Bereich von knapp unter 600 cm À1 erwarten (SI) und ist damit als Ergänzung zu der von Pelmentschikov,C ramer und Mitarbeitern vorgeschlagenen Signatur bei 226 cm À1 fürd ie weitere Analyse der prosthetischen Gruppe der Nitrogenase während der Katalyse zu sehen. [19] Auch die Festigkeit innerhalb des Eisen-Clusters selbst ist nach unseren Berechnungen deutlich rigider als bisher angenommen:M it einer Fe-Fe-Kraftkonstante von 1.23 Ncm À1 (empirische NRVS-Kraftkonstante:0 .27 Ncm À1 ; siehe Tabelle 1) müssen auch diese Bindungen deutlich rigider als bisher angenommen werden. Sie sind damit zwar immer noch weit vom Wert einer Fe-Fe-Zwei-Elektronen-Bindung entfernt (deutlich über 2Ncm À1 ), [20] aber im Einklang mit der schon sehr frühv on Deng und Hoffmann vermuteten Kovalenz im FeMo-Cofaktor.…”